TLR 9 inhibitors

ABSTRACT

A method for inhibiting TLR9 includes contacting the TLR9 with compound of general formula (I): 
                         
wherein the meanings of the variables are explained in the specification,
 
or a stereoisomeric form or a mixture of stereoisomeric forms, or pharmaceutically acceptable salts thereof. A pharmaceutical composition can include compounds of the invention, which can be used in a method for inhibiting TLR9 activity in vitro or in vivo. The method can be performed by administering the compound to a subject to inhibit TLR9 activity, which can be used to treat a disease or disorder associated with TLR9.

CROSS-REFERENCE

This patent application is a continuation-in-part of U.S. applicationSer. No. 16/861,142 filed Apr. 28, 2020, which is a divisional of U.S.application Ser. No. 16/262,631 filed Jan. 30, 2019, which applicationsare incorporated herein by specific reference in their entirety.

BACKGROUND Technical Field

The present invention relates to novel compounds, which are useful asToll-like receptor (“TLR”) inhibitors, and in particular, TLR9inhibitors. Provided herein are novel compounds, compositions comprisingsuch compounds, method for preparation thereof and methods of their use.The invention relates to pharmaceutical compositions containing at leastone compound according to the invention that are useful for thetreatment of conditions related to TLR9 inhibition, such as inflammatoryand/or autoimmune diseases, and methods of inhibiting the activity ofTLR9 in a subject.

Background Art

Toll-like receptors (TLRs) represent transmembrane proteins that detectinvading pathogens by binding pathogen derived molecules and that inducesignaling cascades for proinflammatory gene expression. More precisely,TLRs recognize highly conserved structural motifs known aspathogen-associated microbial patterns (PAMPs), which are exclusivelyexpressed by microbial pathogens, or danger-associated molecularpatterns (DAMPs) that are endogenous molecules released from necrotic ordying cells. This includes intracellular proteins such as heat shockproteins as well as protein fragments from the extracellular matrix(McCarthy C. et al, “Toll-like receptors and damage-associated molecularpatterns: novel links between inflammation and hypertension” Am. J.Physiol. Heart. Circ. Physiol., 2014, 15 January; 306(2):H184-96).

The TLRs were reported as a key component of innate and adaptiveimmunity (Pasare C., et al (2005) “Toll-Like Receptors: Linking Innateand Adaptive Immunity”. In: Gupta S., Paul W. E., Steinman R. (eds)Mechanisms of Lymphocyte Activation and Immune Regulation X. Advances inExperimental Medicine and Biology, vol 560. Springer, Boston, Mass.).

Upon PAMP recognition, the TLR typically induces intracellular signalingcascades. An inflammatory response for a short duration can bebeneficial because it helps to clear the infectious agent. However,prolonged inflammation is not desirable due to possible tissue damage.Indeed, excessive production of inflammatory cytokines and chemokinesvia TLR pathways is often associated with many inflammatory-associatedand autoimmune diseases. Therefore, fine control of inflammation in theTLR pathway is highly desirable for effective host defense.

TLR9 are expressed in immune system cells, such as dendritic cells,macrophages, natural killer cells, and other antigen presenting cells.The TLR9 preferentially binds DNA present in bacteria and viruses, andtriggers signaling cascades that lead to pro-inflammatory cytokineresponses. Additionally, cancer, infection, and tissue damage can allmodulate TLR9 expression and activity. TLR9 is a factor in autoimmunediseases, where TLR9 antagonists can help regulate autoimmuneinflammation.

Additionally, TLR9 expression may be higher in breast cancer, ovariancancer, prostate cancer, non-small cell lung cancer, and glioma.Accordingly, inhibiting TLR9 may be used to inhibit these cancers orothers. TLR9 may play a role in non-viral cancers

TLR9 antagonists can be used to inhibit TLR9 recognition of specificunmethylated CpG oligonucleotides (ODN) that distinguish microbial DNAfrom mammalian DNA. As such, TLR9 antagonists can neutralize thestimulatory effect of CpG ODNs. This inhibition can inhibit theinflammatory response or associated inflammatory disorder. A TLR9antagonist can be used as a therapeutic agent for CpG-ODN-mediatedover-inflammatory responses, and may also be used to treat autoimmunediseases.

Previously, some TLR9 inhibitors have been known to be unselectivebecause they inhibit multiple types of TLRs. For example, WO2020/020800teaches compounds that inhibit TLR7, TLR8, and TLR9. Additionally,WO2008/152471 teaches another type of compound that also inhibits TLR7,TLR8, and TLR9. As a result, the compounds recited in these referencecannot selectively inhibit TLR9.

TLRs are expressed on many types of cancer cells. During chronicinflammation, abnormal activation of TLRs in normal fibroblasts andepithelial cells might facilitate neoplastic transformation andcarcinogenesis. Cancer cells activated by TLR signals can releasecytokines and chemokines that recruit and optimize immune cells torelease further cytokines and chemokines. The result is an aberrantcytokine profile associated with immune tolerance, cancer progressionand propagation of the tumor microenvironment (Sato Y. et al, “CancerCells Expressing Toll-like Receptors and the Tumor” Cancer Microenviron.2009 September; 2(Suppl 1): 205-214).

Concluding, excessive TLR activation can affect the immune systemhomeostasis by excessive pro-inflammatory cytokines and chemokinesproduction, and consequently is responsible for the development of manyinflammatory and autoimmune diseases, such as systemic lupus,infection-associated sepsis, atherosclerosis, and asthma, and cancerdeceases. It is therefore believed that inhibitors/antagonists targetingTLR signals may be beneficial to treat these disorders.

Thus, it is desirable to regulate the pro-inflammatory andanti-inflammatory cytokines and chemokines in the TLR-mediated pathways.Therefore, the technical problem to be solved by the present inventionis to provide a new type of effective TLR9 inhibitor that can be usedduring treatment of, inter alia, autoimmune deceases, inflammatorydiseases and cancer diseases.

SUMMARY

In some embodiments, a method for inhibiting TLR9 activity, the methodcomprising: administering a compound to a TLR9 in an amount sufficientto inhibit activity thereof, the compound having a structure of Formula(I), or a stereoisomeric form, a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof,

wherein,

ring B is a substituted monocycle containing 3-7 atoms, the monocyclebeing selected from an aryl or heteroaryl, wherein the heteroaryl hasfrom 1 to 4 heteroatoms, which are independently selected from nitrogen,oxygen, and sulfur;

G represents a substituted or unsubstituted C₀-C₅ alkylene;

one of W, U, E and J represents CR-T and the rest of W, U, and J areindependently absent or independently represent CR₂ or NR; T represents:

wherein,

Z is an amide in either orientation, which can be selected from:

X represents (—CH₂—)_(n) wherein n=1 to 12, thereby forming an alkylenechain, wherein the alkylene chain is optionally substituted withhalogen, C₁-C₂₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, halogenatedC₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, or C₁-C₁₀alkoxy;

A is 3 to 8 membered heterocyclyl fused with an aryl or heteroaryl,which can be unsubstituted or substituted with one or more Rsubstituents;

R₁ is one or more of, independently of each other, H, halogen,halogenated C₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, or —CN;

R₂ is one or more of, independently of each other, H, C₁-C₂₀ alkyl,halogenated C₁-C₂₀ alkyl, —OR, —SR, or —CN; and

each R is independently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl,hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, halogenated C₁-C₂₀ alkyl, halogen,—OH, —NO₂, —CN, —COOH, —CHO, —SO₃H, —SO₂R, —SOR, —NH₂, —NHR, —NR₂,CHal₃, —NHCO(C₁-C₁₀)alkyl, —CONHR, —C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R,—NRC(O)N(R)₂, —NRSO₂R.

In some embodiments, the method includes inhibiting TLR9 activity withthe compound.

In some embodiments, the method includes contacting cells with thecompound, wherein the cells express the TLR9.

In some embodiments, the compound is administered to the TLR9 in vitro.

In some embodiments, the compound is administered to the TLR9 in vivo.

In some embodiments, the compound is administered to a subject havingthe TLR9, wherein the subject is susceptible or has a disease ordisorder mediated by the TLR9. In some aspects, the subject has aincludes at least one of: a disorder or disease associated with theover-stimulation of the subject's immune system by microbes;interferon-mediated diseases; or inflammatory cytokine-mediatedinflammation diseases. In some aspects, the method includes treating thedisorder or disease associated with the over-stimulation of thesubject's immune system by microbes. In some aspects, the methodincludes treating the interferon-mediated disease. In some aspects, themethod includes treating the inflammatory cytokine-mediated inflammationdiseases. In some aspects, the method includes treating at least one of:antiphospholipid syndrome, autoimmune hepatitis, autoimmune myocarditis,autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy,rheumatoid arthritis, psoriatic arthritis, osteoarthritis, systemicLupus Erythematosus, lupus nephritis, osteoporosis, systemic sclerosis,multiple sclerosis, psoriasis, diabetes, inflammatory bowel disease(Crohn's Disease and Ulcerative Colitis), Hyperimmunoglobulinemia D,periodic fever syndrome, systemic juvenile idiopathic arthritis, sepsis,atherosclerosis, Celiac disease, Sjogren's Syndrome, Alzheimer'sdisease, Parkinson's disease, or cancer. In some aspects, the cancer isselected from colorectal cancer, breast cancer, ovarian carcinoma,pancreatic cancer, lung cancer, renal cell carcinoma, cervical cancerand multiple myeloma. In some aspects, the method includes inhibitinginflammation in the subject with the compound. In some aspects, themethod includes inhibiting activation of an immune system of the subjectwith the compound.

In some embodiments, the compounds are selective for TLR9. In someinstances, the selectivity for TLR9 is compared to a TLR7 or TLR8. Insome instances, the selectivity for TLR9 is compared to other receptors.In some instances, the selectivity for TLR9 is compared to other TLRs.Thus, the compounds can selectivity target and inhibit a TLR9 over othertypes of receptors.

In some embodiments, the method is performed with a compound having astructure of Formula (III), or a stereoisomeric form, a mixture ofstereoisomeric forms, or pharmaceutically acceptable salts thereof,

wherein,

Y and L are independently CR or CH,

one of W, U, E and J represents CH(T) and the rest of W, U, and J areindependently absent or independently represent CH₂;

T represents:

wherein,

Z is an amide in either orientation, which can be selected from:

X represents (—CH₂—)_(n) wherein n=1 to 24, thereby forming an alkylenechain, wherein the alkylene chain is optionally substituted withhalogen, C₁-C₂₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, halogenatedC₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, or C₁-C₁₀alkoxy;

A represents:

any one of A1, A2, A3, or A4 is unsubstituted or substituted with one ormore R groups;

R₁ is one or more of, independently of each other, H, halogen,halogenated C₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, or —CN;

R₂ is one or more of, independently of each other, H, C₁-C₂₀ alkyl,halogenated C₁-C₂₀ alkyl, —OR, —SR, or —CN; and

each R is independently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl,hydroxy C₁-C₁₀ alkyl, alkoxy, halogenated C₁-C₂₀ alkyl, halogen, —OH,—NO₂, —CN, —COOH, —CHO, —SO₃H, —SO₂R, —SOR, —NH₂, —NHR, —NR₂, CHal₃,—NHCO(C₁-C₁₀)alkyl (e.g., alkyl-amide), —CONHR, —C(O)R, —CO₂R,—C(O)N(R)₂, —NRC(O)R, —NRC(O)N(R)₂, —NRSO₂R.

In some aspects, Y is CR and L is CH, R₁ is para position;

one of W, U, E and J represents CH(T) and the rest of W, U, and J areindependently absent or independently represent CH₂;

X represents (—CH₂—)_(n) wherein n=1, 2, or 3, thereby forming analkylene chain;

A represents:

A1 is unsubstituted or substituted with one or more R groups;

R₁ is one or more of, independently of each other, H, halogen,halogenated C₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, or —CN;

R₂ is one or more of, independently of each other, H, C₁-C₂₀ alkyl,halogenated C₁-C₂₀ alkyl, —OR, —SR, or —CN; and

each R is independently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl,hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, halogenated C₁-C₂₀ alkyl, halogen,—OH, —NO₂, —CN.

In some embodiments, the compound has a structure of Formula (IV), or astereoisomeric form, a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof,

wherein,

Y and L are independently CH or CR, wherein at least one of Y or L isCH;

G1 is CH₂ or absent;

each R is independently selected from H, halogen, C₁-C₆ alkyl,halogenated C₁-C₆ alkyl, hydroxy C₁-C₆ alkyl, C₁-C₆ alkoxy, or —CN; and

X represents (—CH₂—)_(n), wherein n=2 or 3, thereby forming an alkylenechain.

In some embodiments, the compound has a structure of Formula (VI), or astereoisomeric form, a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof,

wherein,

G1 is CH or absent;

X represents (—CH₂—)_(n) wherein n=1 to 4, thereby forming an alkylenechain, the alkylene chain is optionally substituted with halogen, C₁-C₆alkyl, C₂-C₈ alkenyl, C₃-C₈ cycloalkyl, C₂-C₈ alkynyl, halogenated C₁-C₆alkyl, hydroxy C₁-C₆ alkyl, or C₁-C₆alkoxy;

R3 is one or more of H, halogen, halogenated C₁-C₁₀ alkyl, hydroxyC₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, or —CN;

R4 is one or more of H, halogen, halogenated C₁-C₁₀ alkyl, hydroxyC₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, or —CN;

R5 is one or more of H, halogen, halogenated C₁-C₁₀ alkyl, hydroxyC₁-C₁₀ alkyl, or C₁-C₁₀ alkoxy.

In some aspects: G1 is CH or absent; X represents (—CH₂—)_(n) whereinn=2 or 3, thereby forming an alkylene chain; R3 is —CN; R4 is one ormore of H, halogen, or halogenated C₁-C₁₀ alkyl; and R5 is one or moreof H, hydroxy C₁-C₁₀ alkyl, or C₁-C₁₀ alkoxy.

In some embodiments, the compound is selected from:

In some embodiments, the compound is selected from: Compound 1, Compound4, Compound 9, or Compound 25.

DETAILED DESCRIPTION

The present invention relates to compounds of general Formula (I)possessing properties of TLR9 inhibition.

In a first aspect, the invention relates to a compound of generalFormula (I):

wherein,ring B is a substituted or unsubstituted monocycle containing 3-7 atoms,the monocycle being selected from a cycloalkyl, aryl, heterocyclyl orheteroaryl,wherein the heterocyclyl or heteroaryl has from 1 to 4 heteroatoms,which are independently selected from nitrogen, oxygen, and sulfur;G represents a substituted or unsubstituted C₀-C₅ alkylene;one of W, U, E and J represents CR-T or N-T and the rest of W, U, and Jare independently absent or independently represent CR₂, NR or S;T represents:

wherein,Z is selected from —O—C(O)—, —(O)C—O—, —N—C(O)—, —(O)C—N—, —O—C(NR)—,—(NR)C—O—, —O—C(S)—, —(S)C—O—, —C(O)—, —C(O)ON—, and —N—C(O)—O—;X represents (—CH₂—)_(n) wherein n=1 to 24, thereby forming an alkylenechain, wherein the carbon atoms of the alkylene chain can be replaced byat least one heteroatom, wherein the heteroatoms are independently —O—,—S— or —NH—, with the proviso that each heteroatom is separated fromeach other heteroatom by at least one carbon atom; the alkylene chain isoptionally substituted with halogen, C₁-C₂₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, halogenated C₁-C₁₀ alkyl, hydroxy alkyl, orC₁-C₁₀alkoxy;A is 3 to 8 membered cycloalkyl, heterocyclyl, aryl or heteroaryl, whichcan be unsubstituted or substituted with one or more R substituents,R₁ is one or more of, independently of each other, H, C₁-C₂₀ alkyl,C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, halogen, halogenated C₁-C₁₀ alkyl,hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, —CN, a 3-8 membered saturated orpartially unsaturated carbocyclic ring, a 3-7 membered heterocylic ringhaving 1-4 heteroatoms that are independently selected from nitrogen,oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having1-4 heteroatoms that are independently selected from nitrogen, oxygen,or sulfur;R₂ is one or more of, independently of each other, H, C₁-C₂₀ alkyl,halogenated C₁-C₂₀ alkyl, —OR, —SR, —CN, a 3-8 membered saturated orpartially unsaturated carbocyclic ring, a 3-7 membered heterocylic ringhaving 1-4 heteroatoms that are independently selected from nitrogen,oxygen, or sulfur, or a 5-6 membered monocyclic heteroaryl ring having1-4 heteroatoms that are independently selected from nitrogen, oxygen,or sulfur; andeach R is independently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl,hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, halogenated C₁-C₂₀ alkyl, halogen,—OH, —NO₂, —CN, —COOH, —CHO, —SO₃H, —SO₂R, —SOR, —NH₂, —NHR, —NR₂,CHal₃, —NHCO(C₁-C₁₀)alkyl (e.g., alkyl-amide), —CONHR, —C(O)R, —CO₂R,—C(O)N(R)₂, —NRC(O)R, —NRC(O)N(R)₂, —NRSO₂R; a 3-8 membered saturated orpartially unsaturated cycloalkyl, C₃₋₁₀ aryl, a 3-7 memberedheterocyclic ring having 1-4 heteroatoms that are independently selectedfrom nitrogen, oxygen, or sulfur, or a 5-6 membered heteroaryl having1-4 heteroatoms that are independently selected from nitrogen, oxygen,or sulfur, or two R groups together, if present, form 3-8 memberedsaturated or unsaturated carbocyclic or heterocyclic ring which containsat least one heteroatom selected from N, S and O, or a stereoisomericform or a mixture of stereoisomeric forms, or pharmaceuticallyacceptable salts thereof.

Preferably, both ring B and the ring formed by N, Y, W, E, J and C aremonocycle rings, i.e. they are not condensed with other saturated orunsaturated rings.

With regard to Formula I, the TLR9 antagonist can include:

ring B is a substituted monocycle containing 3-7 atoms, the monocyclebeing selected from an aryl or heteroaryl, wherein the heteroaryl hasfrom 1 to 4 heteroatoms, which are independently selected from nitrogen,oxygen, and sulfur;

G represents a substituted or unsubstituted C₀-C₅ alkylene;

one of W, U, E and J represents CR-T and the rest of W, U, and J areindependently absent or independently represent CR₂ or NR;

T represents:

wherein,Z is selected from —O—C(NR)— or —(NR)C—O—;X represents (—CH₂—)_(n) wherein n=1 to 24, thereby forming an alkylenechain, wherein the alkylene chain is optionally substituted withhalogen, C₁-C₂₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, halogenatedC₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, or C₁-C₁₀alkoxy;A is 3 to 8 membered heterocyclyl fused with an aryl or heteroaryl,which can be unsubstituted or substituted with one or more Rsubstituents;R₁ is one or more of, independently of each other, H, halogen,halogenated C₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, or —CN;R₂ is one or more of, independently of each other, H, C₁-C₂₀ alkyl,halogenated C₁-C₂₀ alkyl, —OR, —SR, or —CN; andeach R is independently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl,hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, halogenated C₁-C₂₀ alkyl, halogen,—OH, —NO₂, —CN, —COOH, —CHO, —SO₃H, —SO₂R, —SOR, —NH₂, —NHR, —NR₂,CHal₃, —NHCO(C₁-C₁₀)alkyl (e.g., alkyl-amide), —CONHR, —C(O)R, —CO₂R,—C(O)N(R)₂, —NRC(O)R, —NRC(O)N(R)₂, —NRSO₂R;or a stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

In the second aspect, the invention relates to a compound of formula (I)characterized in that the compound is a compound of general formula(II):

wherein,Y and L are independently CR or N; or one of Y and L is absent,one of W, U, E and J represents —CH(T)- or N-T and the rest of W, U, Eand J are independently absent or independently represent CR₂, NR or S;G represents an unsubstituted C₀-C₅ alkylene;

-   -   T represents:

wherein,Z is selected from —O—C(O)—, —(O)C—O—, —N—C(O)—, —(O)C—N—, —O—C(NR)—,—(NR)C—O—, —O—C(S)—, —(S)C—O—, —C(O)—, —C(O)ON—, and —N—C(O)—O—;X represents (—CH₂—)_(n) wherein n=1 to 12, thereby forming an alkylenechain, wherein the carbon atoms of the alkylene chain can be replaced byat least one heteroatom, wherein the heteroatoms are independently —O—,—S— or —NH—, with the proviso that each heteroatom is separated fromeach other heteroatom by at least one carbon atom; the alkylene chain isoptionally substituted with a halogen, C₁-C₂₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, halogenated C₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, orC₁-C₁₀alkoxy;A is 3-8 membered substituted or unsubstituted cycloalkyl, heterocyclyl,aryl or heteroaryl,R₁ is one or more of, independently of each other, H, C₁-C₁₂ alkyl,C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, F, Cl, halogenated C₁-C₄ alkyl, hydroxyC₁-C₄ alkyl, C₁-C₄ alkoxy, or —CN;R₂ is one or more of, independently of each other, H, C₁-C₂₀ alkyl,halogenated C₁-C₂₀ alkyl, —OR, —SR, or —CN; andeach R is independently H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, halogenated C₁-C₂₀ alkyl, halogen,—OH, —NO₂, —CN, —COOH, —CHO, —SO₃H, —SO₂R, —SOR, —NH₂, —NHR, —NR₂,—CHal₃, —NHCO(C₁-C₁₀)alkyl (alkyl-amide), —CONHR, —C(O)R, —CO₂R,—C(O)N(R)₂, —NRC(O)R, —NRC(O)N(R)₂, —NRSO₂R; a 3-8 membered saturated orpartially unsaturated cycloalkyl, C₃₋₁₀ aryl, a 3-7 membered heterocylicring having 1-4 heteroatoms that are independently selected fromnitrogen, oxygen, or sulfur, or a 5-6 membered heteroaryl having 1-4heteroatoms that are independently selected from nitrogen, oxygen, orsulfur, or two R groups together, if present, form 3-8 memberedsaturated or unsaturated carbocyclic or heterocyclic ring which containsat least one heteroatom selected from N, S and O;or a stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

With regard to Formula II, the TLR9 antagonist can include:

Y and L are independently CR or N,

G represents a substituted or unsubstituted C₀-C₅ alkylene;

one of W, U, E and J represents CH(T) and the rest of W, U, and J areindependently absent or independently represent CR₂ or NR;

X represents (—CH₂—)_(n) wherein n=1 to 24, thereby forming an alkylenechain, wherein the alkylene chain is optionally substituted withhalogen, C₁-C₂₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, halogenatedC₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, or C₁-C₁₀alkoxy;

A is 3 to 8 membered heterocyclyl fused with an aryl or heteroaryl,which can be unsubstituted or substituted with one or more Rsubstituents,

R₁ is one or more of, independently of each other, H, halogen,halogenated C₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, or —CN;

R₂ is one or more of, independently of each other, H, C₁-C₂₀ alkyl,halogenated C₁-C₂₀ alkyl, —OR, —SR, or —CN; and

each R is independently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl,hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, halogenated C₁-C₂₀ alkyl, halogen,—OH, —NO₂, —CN, —COOH, —CHO, —SO₃H, —SO₂R, —SOR, —NH₂, —NHR, —NR₂,CHal₃, —NHCO(C₁-C₁₀)alkyl (e.g., alkyl-amide), —CONHR, —C(O)R, —CO₂R,—C(O)N(R)₂, —NRC(O)R, —NRC(O)N(R)₂, —NRSO₂R;or a stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

In further aspect, the compound of the invention is characterized inthat the compound is a compound of general formula (III):

wherein,Y and L are independently CR or N; or optionally one of Y and L isabsent,one of W, U, E and J represents —CH(T)- or N-T and the rest of W, U, Eand J are independently absent or independently represent CR₂, NR or S;G represents an unsubstituted C₀-C₅ alkylene;T represents:

wherein,X represents —(CH₂—)_(n) wherein n=1 to 6, thereby forming an alkylenechain, wherein the carbon atoms of the alkylene chain can be replaced byat least one heteroatom, wherein the heteroatoms are independently —O—,—S— or —NH—, with the proviso that each heteroatom is separated fromeach other heteroatom by at least one carbon atom; the alkylene chain isoptionally substituted with a halogen, C₁-C₂₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, halogenated C₁-C₁₀ alkyl, hydroxy C₁-C₁₀alkyl; orC₁-C₁₀alkoxy;A is 3 to 8 membered substituted or unsubstituted cycloalkyl,heterocyclyl, aryl or heteroaryl,R₁ is one or more of, independently of each other, H, C₁-C₁₂ alkyl,C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, F, Cl, halogenated C₁-C₄alkyl, hydroxyC₁-C₄alkyl, C₁-C₄alkoxy, or —CN;R₂ is one or more of, independently of each other, H, C₁-C₂₀ alkyl,halogenated C₁-C₂₀ alkyl, —OR, —SR, or —CN; andeach R is independently H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, halogenated C₁-C₂₀ alkyl, halogen,—OH, —NO₂, —CN, —COOH, —CHO, —SO₃H, —SO₂R, —SOR, —NH₂, —NHR, —NR₂,—CHal₃, —NHCO(C₁-C₁₀)alkyl, —CONHR, —C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R,—NRC(O)N(R)₂, —NRSO₂R; a 3-8 membered saturated or partially unsaturatedcycloalkyl, C₃₋₁₀ aryl, a 3-7 membered heterocylic ring having 1-4heteroatoms that are independently selected from nitrogen, oxygen, orsulfur, or a 5-6 membered heteroaryl having 1-4 heteroatoms that areindependently selected from nitrogen, oxygen, or sulfur, or two R groupstogether, if present, form 3-8 membered saturated or unsaturatedcarbocyclic or heterocyclic ring which contains at least one heteroatomselected from N, S and O;or a stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

With regard to Formula III, the TLR9 antagonist can include:

Y and L are independently CR or CH,

one of W, U, E and J represents CH(T) and the rest of W, U, and J areindependently absent or independently represent CH₂;

X represents (—CH₂—)_(n) wherein n=1 to 24, thereby forming an alkylenechain, wherein the alkylene chain is optionally substituted withhalogen, C₁-C₂₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, halogenatedC₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, or C₁-C₁₀alkoxy;

A is 3 to 8 membered heterocyclyl fused with an aryl or heteroaryl,which can be unsubstituted or substituted with one or more Rsubstituents,

R₁ is one or more of, independently of each other, H, halogen,halogenated C₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, or —CN;

R₂ is one or more of, independently of each other, H, C₁-C₂₀ alkyl,halogenated C₁-C₂₀ alkyl, —OR, —SR, or —CN; and

each R is independently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl,hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, halogenated C₁-C₂₀ alkyl, halogen,—OH, —NO₂, —CN, —COOH, —CHO, —SO₃H, —SO₂R, —SOR, —NH₂, —NHR, —NR₂,CHal₃, —NHCO(C₁-C₁₀)alkyl (e.g., alkyl-amide), —CONHR, —C(O)R, —CO₂R,—C(O)N(R)₂, —NRC(O)R, —NRC(O)N(R)₂, —NRSO₂R;or a stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

With regard to Formula III, the TLR9 antagonist can include:

Y and L are independently CR or CH,

one of W, U, E and J represents CH(T) and the rest of W, U, and J areindependently absent or independently represent CH₂;

X represents (—CH₂—)_(n) wherein n=1 to 24, thereby forming an alkylenechain, wherein the alkylene chain is optionally substituted withhalogen, C₁-C₂₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, halogenatedC₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, or C₁-C₁₀alkoxy;

A is 3 to 8 membered heterocyclyl fused with an aryl or heteroaryl,which can be unsubstituted or substituted with one or more Rsubstituents,

R₁ is one or more of, independently of each other, H, halogen,halogenated C₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, or —CN;

R₂ is one or more of, independently of each other, H, C₁-C₂₀ alkyl,halogenated C₁-C₂₀ alkyl, —OR, —SR, or —CN; and

each R is independently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl,hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, halogenated C₁-C₂₀ alkyl, halogen,—OH, —NO₂, —CN, —COOH, —CHO, —SO₃H, —SO₂R, —SOR, —NH₂, —NHR, —NR₂,CHal₃, —NHCO(C₁-C₁₀)alkyl (e.g., alkyl-amide), —CONHR, —C(O)R, —CO₂R,—C(O)N(R)₂, —NRC(O)R, —NRC(O)N(R)₂, —NRSO₂R;or a stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

In yet another aspect, the invention relates to a compound of formula(III) as defined herein, wherein in the formula (III) with thedefinitions provided herein, the T represents:

or a stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

In a further aspect of the present invention, in the formula (III), asdefined herein, the T represents:

wherein any of these structures can be unsubstituted or substituted withone or more R groups,

each R is independently H, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl,hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, halogenated C₁-C₂₀ alkyl, halogen,—OH, —NO₂, —CN, —COOH, —CHO, —SO₃H, —SO₂R, —SOR, —NH₂, —NHR, —NR₂,—CHal₃, —NHCO(C₁-C₁₀)alkyl, —CONHR, —C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R,—NRC(O)N(R)₂, —NRSO₂R; a 3-8 membered saturated or partially unsaturatedcycloalkyl, C₃₋₁₀ aryl, a 3-7 membered heterocylic ring having 1-4heteroatoms that are independently selected from nitrogen, oxygen, orsulfur, or a 5-6 membered heteroaryl having 1-4 heteroatoms that areindependently selected from nitrogen, oxygen, or sulfur;

or a stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

In some embodiments of the TLR9 inhibitor, for the formula I-III, T isT5 with Z being 0 and X represents (—CH₂—)_(n) wherein n=1 to 24,thereby forming an alkylene chain, wherein the alkylene chain isoptionally substituted with halogen, C₁-C₂₀ alkyl, C₂-C₁₀ alkenyl,C₂-C₁₀ alkynyl, halogenated C₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, orC₁-C₁₀alkoxy.

Further, the invention relates to a compound of formula (III) as definedherein, wherein one of W, U, E and J represents —CH(T)- or N-T and therest of W, U, E and J are independently absent or independentlyrepresent CH₂, CR₂, NR or S; and T represents:

wherein X is —(CH₂)_(n)— and n is 1 to 5,A is 3-8 membered substituted or unsubstituted cycloalkyl, heterocyclyl,aryl or heteroaryl,or A is 3 to 8 membered heterocyclyl fused with an aryl or heteroaryl,which can be unsubstituted or substituted with one or more Rsubstituents; ora stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

In yet another aspect of the present invention, in the formula (III),one of W, U, E and J represents —CH(T)- or N-T and the rest of W, U, Eand J are independently absent or independently represent CH₂, CR₂, NRor S; T represents:

wherein the rings in T4, T5, T6, T7, T8, T9, T10, T11, or T12 areunsubstituted or substituted with one or more R groups,wherein each R is independently H, C1-C10 alkyl, C2-C10 alkenyl, C2-C10alkynyl, hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, halogenated C₁-C₂₀ alkyl,halogen, —OH, —NO₂, —CN, —COOH, —CHO, —SO₃H, —SO₂R, —SOR, —NH₂, —NHR,—NR₂, —CHal₃, —NHCO(C₁-C₁₀)alkyl, —CONHR, —C(O)R, —CO₂R, —C(O)N(R)₂,—NRC(O)R, —NRC(O)N(R)₂, —NRSO₂R; a 3-8 membered saturated or partiallyunsaturated cycloalkyl, C₃₋₁₀ aryl, a 3-7 membered heterocylic ringhaving 1-4 heteroatoms that are independently selected from nitrogen,oxygen, or sulfur, or a 5-6 membered heteroaryl having 1-4 heteroatomsthat are independently selected from nitrogen, oxygen, or sulfur;or a stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

Further the invention relates to a compound of formula (III) as definedherein, wherein one of W, U, E and J represents —CH(T)- or N-T and therest of W, U, E and J are independently absent or independentlyrepresent CH₂, CR₂, NR or S; X in the T structures represents(—CH₂—)_(n) wherein n=1 to 6, thereby forming an alkylene chain, whereinthe carbon atoms of the alkylene chain can be replaced by at least oneheteroatom, wherein the heteroatoms are independently —O—, or —NH—, withthe proviso that each heteroatom is separated from each other heteroatomby at least one carbon atom; the alkylene chain is optionallysubstituted with a halogen, C₁-C₆ alkyl, C₂-C₈ alkenyl, C₃-C₈cycloalkyl, C₂-C₈ alkynyl, halogenated C₁-C₆ alkyl, hydroxy C₁-C₆alkyl,or C₁-C₆ alkoxy;

A represents a fragment or structure selected from:

in which the structures for A can be unsubstituted or substituted withone or more R groups that are independently selected from C₁-C₆ alkyl,—F, —Cl, —CHF₂, —CF₃, —OMe, —OEt, hydroxy C₁-C₄ alkyl, —OH, or —CN;or a stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

In yet another aspect of the present invention, in the compound offormula (III) as defined herein, the R₁ and R₂ are independentlyselected from halogen, —CN, C₁-C₁₀ alkoxy (—OC₁-C₁₀alkyl), —CHal₃,—C(O)OR, wherein R is H, C₁-C₁₀ alkyl, NR₂, wherein R is independently Hor C₁-C₁₀ alkyl, or two R groups together can form a 3-8 memberedsaturated or unsaturated carbocyclic or heterocyclic ring which containsat least one heteroatom selected from N, S and O,

or a stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

In yet another aspect, in the formula (III) as defined herein, wherein Arepresents:

wherein the number of R groups is varied from 1 to 3, and each R isindependently selected from halogen, C₁-C₆ alkyl, C₂-C₈ alkenyl, C₃-C₈cycloalkyl, C₂-C₈ alkynyl, halogenated C₁-C₆ alkyl, hydroxy C₁-C₆ alkyl,or C₁-C₆ alkoxy,or a stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

In yet another aspect of the present invention, the compound is acompound of formula (IV):

wherein,Y and L are independently CH or N; or optionally one of Y and L isabsent,G1 is CH₂ or absent,each R is independently selected from H, halogen, C₁-C₆ alkyl, C₃-C₈cycloalkyl, halogenated C₁-C₆ alkyl, hydroxy C₁-C₆ alkyl, C₁-C₆alkoxy,or —CN;X represents (—CH₂—)_(n) wherein n=1 to 6, thereby forming an alkylenechain, the alkylene chain is optionally substituted with halogen, C₁-C₆alkyl, C₂-C₈ alkenyl, C₃-C₈ cycloalkyl, C₂-C₈ alkynyl, halogenatedC₁-C₆alkyl, hydroxy C₁-C₆ alkyl, or C₁-C₆ alkoxy;or a stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

In some embodiments, the TLR9 inhibitor of Formula IV can include: Y andL are independently CR or CH, G1 is CH₂ or absent, each R isindependently selected from H, halogen, halogenated C₁-C₆ alkyl, hydroxyC₁-C₆ alkyl, C₁-C₆alkoxy, or —CN, X represents (—CH₂—)_(n) wherein n=1to 6, thereby forming an alkylene chain, or a stereoisomeric form or amixture of stereoisomeric forms, or pharmaceutically acceptable saltsthereof.

In yet another aspect of the present invention, the compound is acompound of formula (V):

whereinY is CH or N; or Y is absent,each R is independently selected from H, halogen, C₁-C₆ alkyl, C₃-C₈cycloalkyl, halogenated C₁-C₆ alkyl, hydroxy C₁-C₆ alkyl, C₁-C₆alkoxy,or —CN;X represents (—CH₂—)_(n) wherein n=1 to 6, thereby forming an alkylenechain, the alkylene chain is optionally substituted with halogen, C₁-C₆alkyl, C₂-C₈ alkenyl, C₃-C₈ cycloalkyl, C₂-C₈ alkynyl, halogenated C₁-C₆alkyl, hydroxy C₁-C₆ alkyl, or C₁-C₆alkoxy;or a stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

In some embodiments, the TLR9 inhibitor can include a compound offormula (VI):

wherein: Y¹ is CH or absent; X¹ represents (—CH₂—)_(n) wherein n=1 to 4,thereby forming an alkylene chain, the alkylene chain is optionallysubstituted with halogen, C₁-C₆ alkyl, C₂-C₈ alkenyl, C₃-C₈ cycloalkyl,C₂-C₈ alkynyl, halogenated C₁-C₆ alkyl, hydroxy C₁-C₆ alkyl, orC₁-C₆alkoxy; R³ is one or more of, independently of each other, H,halogen, halogenated C₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy,or —CN; R⁴ is one or more of, independently of each other, H, halogen,halogenated C₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, or —CN;or a stereoisomeric form or a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof.

In some embodiments of formula (VI), Y¹ is CH or absent; X¹ represents(—CH₂—)_(n) wherein n=2 or 3, thereby forming an alkylene chain; R³ isone or more of, independently of each other, H, halogen, or —CN; R⁴ isone or more of, independently of each other, H, halogen, halogenatedC₁-C₁₀ alkyl; or a stereoisomeric form or a mixture of stereoisomericforms, or pharmaceutically acceptable salts thereof.

In some embodiments of formula (VI), Y¹ is CH or absent; X¹ represents(—CH₂—)_(n) wherein n=2 or 3, thereby forming an alkylene chain; R³ is—CN; R⁴ is H or halogen; or a stereoisomeric form or a mixture ofstereoisomeric forms, or pharmaceutically acceptable salts thereof.

In some embodiments of formula (VI), Y¹ is CH; X¹ represents (—CH₂—)_(n)wherein n=2 or 3, thereby forming an alkylene chain; R³ is —CN; R⁴ is Hor halogen; or a stereoisomeric form or a mixture of stereoisomericforms, or pharmaceutically acceptable salts thereof.

Further the terms as used herein are defined.

The term “alkyl”, as used herein, means a straight-chain (i.e.,unbranched) or branched hydrocarbon chain that is completely saturated.Alkyl groups contain 1-12 carbon atoms. In some embodiments, alkylgroups contain 1-6 carbon atoms. In other embodiments, aliphatic groupscontain 1-4 aliphatic carbon atoms. In still other embodiments, alkylgroups contain 1-3 carbon atoms, and in yet other embodiments, aliphaticgroups contain 1-2 aliphatic carbon atoms.

Exemplary alkyl groups are methyl, ethyl, propyl, isopropyl, butyl,isobutyl, tert-butyl, pentyl, hexyl, heptyl, nonyl and decyl.

The term “cycloalkyl”, as used herein, refers to a monocyclic C₃-C₈hydrocarbon that is completely saturated or that contains one or moreunits of unsaturation, but which is not aromatic, that has a singlepoint of attachment to the rest of the molecule. Exemplary groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl,cycooctyl, cyclodecyl, cyclododecyl and adamantyl.

Unless otherwise indicated, the term “alkenyl” as used herein by itselfor as part of another group refers to straight or branched chainradicals of 2 to 20 carbons, in some embodiments 2 to 12 carbons, and insome embodiments 2 to 8 carbons in the main chain, which include one ormore double bonds in the main chain, such as vinyl, 2-propenyl,3-butenyl, 2-butenyl, 4-pentenyl, 3-pentenyl, 2-hexenyl, 3-hexenyl,2-heptenyl, 3-heptenyl, 4-heptenyl, 3-octenyl, 3-nonenyl, 4-decenyl,3-undecenyl, 4-dodecenyl, 4,8,12-tetradecatrienyl, and the like.“Substituted alkenyl” includes an alkenyl group optionally substitutedwith one or more substituents, such as the substituents included abovein the definition of “alkyl” and “cycloalkyl”.

Unless otherwise indicated, the term “alkynyl” as used herein by itselfor as part of another group refers to straight or branched chainradicals of 2 to 20 carbons, in some embodiments 2 to 12 carbons and insome embodiments 2 to 8 carbons in the normal chain, which include oneor more triple bonds in the normal chain, such as 2-propynyl, 3-butynyl,2-butynyl, 4-pentynyl, 3-pentynyl, 2-hexynyl, 3-hexynyl, 2-heptynyl,3-heptynyl, 4-heptynyl, 3-octynyl, 3-nonynyl, 4-decynyl, 3-undecynyl,4-dodecynyl and the like. “Substituted alkynyl” includes an alkynylgroup optionally substituted with one or more substituents, such as thesubstituents included above in the definition of “alkyl” and“cycloalkyl.”

The term “halogen” means F, Cl, Br, or I.

The terms “halogenated alkyl”, “halogenated alkenyl” and “alkynyl” asused herein alone or as part of another group refers to “alkyl”,“alkenyl” and “alkynyl” which are substituted by one or more atomsselected from fluorine, chlorine, bromine, fluorine, and iodine.

The term “alkoxyl” refers to straight and branched aliphatic hydrocarbonchains attached to an oxygen atom, for example methoxy, ethoxy,n-propoxy, isopropoxy and the like.

The term “heteroatom” means one or more of oxygen, sulfur, nitrogen, orphosphorus (including, any oxidized form of nitrogen, sulfur, orphosphorus; the quaternized form of any basic nitrogen or; asubstitutable nitrogen of a heterocyclic ring, for example N (as in3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR+ (as inN-substituted pyrrolidinyl)).

The term “alkylene” refers to a bivalent alkyl group. An “alkylenechain” is a polymethylene group, i.e., —(CH2)n-, wherein n is a positiveinteger, preferably from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2,or from 2 to 3. A substituted alkylene chain is a polymethylene group inwhich one or more methylene hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “alkenylene” refers to a bivalent alkenyl group. A substitutedalkenylene chain is a polymethylene group containing at least one doublebond in which one or more hydrogen atoms are replaced with asubstituent. Suitable substituents include those described below for asubstituted aliphatic group.

The term “aryl” used individually or as part of a larger moiety as in“aralkyl”, “aralkoxy”, or “aryloxy alkyl”, unless otherwise indicated,refers to monocyclic and bicyclic ring systems having a total of 3 to 14ring members, wherein at least one ring in the system is aromatic andwherein each ring in the system contains three to seven ring members. Incertain embodiments of the present invention, “aryl” refers to anaromatic ring system. Exemplary aryl groups are cyclopentadienyl,phenyl, biphenyl, naphthyl, anthracyl and the like. Also included withinthe scope of the term “aryl”, as it is used herein, is a group in whichan aromatic ring is fused to one or more non-aromatic rings, such asindanyl, phthalimidyl, naphthimidyl, phenanthridinyl, ortetrahydronaphthyl, and the like.

The terms “heteroaryl” used alone or as part of a larger moiety, e.g.,“heteroaralkyl”, or “heteroaralkoxy”, refer to groups having 5 to 10ring atoms, preferably 5, 6, or 9 ring atoms; and having, in addition tocarbon atoms, from one to five heteroatoms. The term “heteroatom” refersto nitrogen, oxygen, or sulfur, and includes any oxidized form ofnitrogen or sulfur, and any quaternized form of a basic nitrogen.

Heteroaryl groups include, without limitation, thienyl, furanyl,pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl,isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl,pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl,naphthyridinyl, and pteridinyl, indolyl, isoindolyl, benzothienyl,benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzthiazolyl,quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl,quinoxalinyl, 4H-quinolizinyl, carbazolyl, acridinyl, phenazinyl,phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, and pyrido[2,3-b]-1,4-oxazin-3(4H)-one.

The term “heteroaralkyl” refers to an alkyl group substituted by aheteroaryl, bonded with alkyl and heteroaryl portions.

The term “heterocyclyl”, unless otherwise indicated, refers to a 3- to7-membered, preferably 5- to 7-membered, monocyclic or 7-10-memberedbicyclic heterocyclic moiety which can be saturated or partiallyunsaturated. In addition to carbon atoms, one or more, preferably one tofour, heteroatoms, can be contained as defined above.

Examples of such saturated or partially unsaturated heterocyclicradicals include, without limitation, tetrahydrofuranyl,tetrahydrothiophenyl pyrrolidinyl, piperidinyl, pyrrolinyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl,thiazepinyl, morpholinyl, and quinuclidinyl. Also within the inventionheterocyclyl ring can be fused to one or more aryl, heteroaryl, orcycloalkyl rings, such as indolinyl, 3H-indolyl, chromanyl,phenanthridinyl, or tetrahydroquinolinyl, where the radical or point ofattachment is on the heterocyclyl ring. A heterocyclyl group isoptionally mono- or bicyclic.

When forming a radical, a heterocyclic ring can be attached to the mainmolecule at any heteroatom or carbon atom that allow to form a stablestructure.

The term “monocyclic” refers to a monovalent saturated or partiallyunsaturated or aromatic cyclic radical having no fused rings attached,but optionally having substituents in any suitable atom within thecycle.

Unless otherwise indicated, an “optionally substituted” group has asuitable substituent at any moiety position available for substitution.Number of substituent is defined by stability of molecule and whilechoosing the substituents the one skilled in the art would easily definewhich character and number of substituents can be used depending on theapplication field. If otherwise indicated, the substituent can beselected from H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl,halogenated C₁-C₂₀ alkyl, halogen, —OH, —NO₂, —CN, —COOH, —CHO, —SO₃H,—SO₂R, —SOR, —NH₂, —NHR, —NR₂, —CHal₃, —NHCO(C₁-C₁₀)alkyl, —CONHR,—C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R, —NRC(O)N(R)₂, —NRSO₂R; a 3-8membered saturated or partially unsaturated cycloalkyl, C₃₋₁₀ aryl, a3-7 membered heterocylic ring having 1-4 heteroatoms independentlyselected from nitrogen, oxygen, or sulfur, or a 5-6 membered heteroarylhaving 1-4 heteroatoms independently selected from nitrogen, oxygen, orsulfur.

As used herein, the term “pharmaceutically acceptable salt” refers tothose salts which are suitable to use within mammals and do not tend tobe toxic. Pharmaceutically acceptable salts are formed using inorganicand organic acids and bases. Examples of pharmaceutically acceptablesalts are salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid, or with organic acids such as tartaric acid, aceticacid, oxalic acid, maleic acid, citric acid, succinic acid or malonicacid, terephthalic acid. Other pharmaceutically acceptable salts includeadipate, ascorbate, aspartate, benzoate, bisulfate, borate, butyrate,valerate, camphorate, camphorsulfonate, cyclopentanepropionate, formate,citrate, oxalate, pivalate, succinate, tartrate, fumarate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactate, laurate, lauryl sulfate, maleate,malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,oleate, palmitate, stearate, undecanoate, alginate, 3-phenylpropionate,phosphate, sulfate, thiocyanate, p-toluenesulfonate, benzenesulfonate,persulfate, ethanesulfonate, dodecylsulfate, and the like and mixturesalts.

Salts derived from appropriate bases include alkali metal, alkalineearth metal, ammonium and N(Calkyl) salts. Representative alkali oralkaline earth metal salts include Sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed using counterions, such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and arylsulfonate.

The compounds of the Formulae I-VI include all possible optical isomersand racemic mixtures thereof. Unless otherwise stated, structuresdepicted herein are meant to include all isomeric (e.g., enantiomeric,diastereomeric, and geometric (or conformational)) forms of thestructure; for example, the R and S configurations for each asymmetriccenter, Z and E double bond isomers, and Z and E conformational isomers.Therefore, single stereo chemical isomers as well as enantiomeric,diastereomeric, and geometric (or conformational) mixtures of thepresent compounds are within the scope of the invention. Unlessotherwise stated, all tautomeric forms of the compounds of the inventionare within the scope of the invention.

The compounds of Formulae I-VI can be used in the form of additionsalts. More particular, acid addition salts can be used such aschlorides, nitrates, sulfates, phosphates, methane sulfonates and saltsof other pharmaceutically acceptable acids. Pharmaceutically acceptableacid-addition salts of compounds of Formula I are generally prepared byreaction of the respective compound with an equimolar amount of arelatively strong acid, preferably an inorganic acid such ashydrochloric, sulfuric or phosphoric acid or an organic acid such asmethanesulfonic acid in a polar solvent. Isolation of the salt isfacilitated by the addition of a solvent in which the salt is insoluble,an example of such a solvent being diethyl ether.

In yet another aspect of the present invention, a pharmaceuticalcomposition is provides comprising one or more compounds as indicatedabove or a salt thereof and a pharmaceutically acceptable carrier ordiluent. More particularly, the pharmaceutical composition according isuseful in the treatment of a disorder or disease which is mediated bythe activity of TLR9, autoimmune diseases and/or inflammatory diseasesand/or cancer. The disorders can be selected from hypersensitivity,diseases associated with the over-stimulation of host's (subject orpatient) immune system by microbes, interferon-mediated diseases orinflammatory cytokine-mediated inflammation diseases.

In another aspect of the present invention, the compound of theinvention is capable of inhibiting TLR9 specifically or preferentiallyover other TLRs. For example, the compounds can inhibit TLR9 over TLR7and TLR8.

In another aspect of the present invention claimed is a pharmaceuticalcomposition comprising one or more compounds of formulae I to VI andspecific embodiments below, or a salt thereof; and a pharmaceuticallyacceptable carrier or diluent.

In another aspect of the present invention claimed is a pharmaceuticalcomposition for use in the treatment of a disorder or disease which ismediated by the activity of TLR9, such as autoimmune diseases and/orinflammatory diseases and/or cancer.

The disorders are selected from diseases associated with theover-stimulation of host's immune system by microbes,interferon-mediated diseases or inflammatory cytokine-mediatedinflammation diseases. Preferably the disorder is selected fromantiphospholipid syndrome, autoimmune hepatitis, autoimmune myocarditis,autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy,rheumatoid arthritis, psoriatic arthritis, osteoarthritis, systemicLupus Erythematosus, lupus nephritis, osteoporosis, systemic sclerosis,multiple sclerosis, psoriasis, diabetes, inflammatory bowel disease(Crohn's Disease and Ulcerative Colitis), Hyperimmunoglobulinemia D,periodic fever syndrome, systemic juvenile idiopathic arthritis, sepsis,atherosclerosis, Celiac disease, Sjogren's Syndrome, Alzheimer'sdisease, Parkinson's disease, and cancer.

Preferably, cancer is selected from colorectal cancer, breast cancer,ovarian carcinoma, pancreatic cancer, lung cancer, renal cell carcinoma,cervical cancer and multiple myeloma.

In another aspect, a method is claimed for inhibiting TLR9 activity in asubject comprising the step of administering to said subject with acompound according to the present invention or a pharmaceuticallyacceptable salt thereof.

The method comprises contacting cells which express the TLR9 in anamount that is sufficient to inhibit the TLR9. The method can bepracticed in vivo or in vitro.

In another embodiment, the invention relates to a method of treating acondition in a patient that is mediated by the binding of TLR9. Themethod comprises administering to the subject a therapeuticallyeffective amount of a compound of the invention. Preferably, thecompound to be administered selectively inhibits the TLR9.

The amount of compound in compositions of this invention is such that itis effective to measurably inhibit TLR9 in a subject.

The term “subject”, as used herein, means an animal, preferably a cell,biological tissue, or animal, preferably mammal, and most preferably ahuman.

The term “pharmaceutically acceptable carrier or pharmaceuticallyacceptable vehicle” refers to a non-toxic carrier, adjuvant, or vehiclethat does not substantially vary the pharmacological activity of thecompound with which it is formulated. Pharmaceutically acceptablecarriers or vehicles that are used in the compositions of this inventioninclude, but are not limited to, lecithin, glycine, sorbic acid,potassium sorbate, sodium chloride, zinc salts, colloidal silica,magnesium trisilicate, polyvinyl pyrrolidone, cellulose-basedsubstances, polyethylene glycol, buffer substances such as cytric acidand phosphates,

A method of the present invention treats disorders are selected fromdiseases associated with the over-stimulation of host's immune system bymicrobes, interferon-mediated diseases or inflammatory cytokine-mediatedinflammation diseases. Preferably, the disease is selected fromantiphospholipid syndrome, autoimmune hepatitis, autoimmune myocarditis,autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy,rheumatoid arthritis, psoriatic arthritis, osteoarthritis, systemicLupus Erythematosus, lupus nephritis, osteoporosis, systemic sclerosis,multiple sclerosis, psoriasis, diabetes, inflammatory bowel disease(Crohn's Disease and Ulcerative Colitis), Hyperimmunoglobulinemia D,periodic fever syndrome, systemic juvenile idiopathic arthritis, sepsis,atherosclerosis, Celiac disease, Sjogren's Syndrome, Alzheimer'sdisease, Parkinson's disease, and cancer, preferably selected fromcolorectal cancer, breast cancer, ovarian carcinoma, pancreatic cancer,lung cancer, renal cell carcinoma, cervical cancer and multiple myeloma.

Compositions of the present invention comprising the compounds ofFormulae (I) to (VI) and specific compounds below described as TLR9inhibitors of the present invention and optionally at least onepharmaceutically acceptable carrier, are acceptable for anyadministration. In particular, they can be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir.

The term “parenteral” as used herein includes subcutaneous, intravenous,intramuscular, etc. Preferably, the compositions are to be administeredorally, or intravenously. Among the acceptable vehicles and solventsthat are employed are water and Ringer's solution, alone or incombination with mono- or di- or poly-glycerides.

Pharmaceutically acceptable compositions of this invention are orallyadministered in any orally acceptable dosage form. Exemplary oral dosageforms are capsules, tablets, aqueous suspensions or solutions. In thecase of tablets for oral use, carriers commonly used include lactose andcorn starch. Lubricating agents, such as magnesium stearate, are alsotypically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch.

Pharmaceutically acceptable compositions of this invention comprisingthe compounds of Formulae (I) to (VI) described as TLR9 inhibitors ofthe present invention are also administered topically, especially whenthe target of treatment includes areas or organs readily accessible bytopical application, including diseases of the eye, the skin, or thelower intestinal tract. Suitable topical formulations are readilyprepared for each of these areas or organs.

For topical applications, provided pharmaceutically acceptablecompositions are formulated in a suitable ointment wherein the compoundof the invention, optionally with other active components, is suspendedor dissolved in one or more carriers. Exemplary carriers for topicaladministration of compounds of this are mineral oil, propylene glycol,polyoxyethylene and water. Suitable topical carriers include, but arenot limited to, mineral oil, sorbitan monostearate, polysorbates, cetylalcohol, benzyl alcohol and water.

Pharmaceutically acceptable compositions of this invention areoptionally administered by nasal aerosol or inhalation. Suchcompositions are prepared according to techniques well-known in the artof pharmaceutical formulation and are prepared as solutions in saline,employing certain conservants, including benzyl alcohol or othersuitable preservatives, and/or other conventional solubilizing ordispersing agents.

The amount of the compounds of Formulae (I) to (VI) described as TLR9inhibitors of the present invention of the present invention that areoptionally combined with the carrier of vehicle materials to produce acomposition in a single dosage form for treating a subject will varydepending upon the host treated, the particular mode of administration.Preferably, provided compositions should be formulated so that a dosageof between 0.01-100 mg/kg body weight/day of the compound can beadministered to a patient receiving these compositions.

The compounds of Formulae (I) to (VI) described as TLR9 inhibitors ofthe present invention can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,capsules, pills, and granules can be prepared with coatings and shellssuch as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms optionally also comprisebuffering agents. They optionally contain opacifying agents and can alsobe of a composition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner. Examples of embedding compositions that can be usedinclude polymeric substances and waxes.

The compounds of Formulae (I) to (VI) described as TLR inhibitors of thepresent invention can may be useful as a within a pharmaceuticalcomposition as a vaccine adjuvant for use in conjunction with anymaterial that modulates immune response, such as, for example, liveviral, bacterial, or parasitic immunogens; inactivated viral,tumor-derived, protozoal, organism-derived, fungal, or bacterialimmunogens, toxoids, toxins; self-antigens; polysaccharides; proteins;glycoproteins; peptides; cellular vaccines; DNA vaccines; recombinantproteins; glycoproteins; peptides; and the like. In some aspects, thecombination therapy including but not limited to the combination of aTLR9 inhibitor and a vaccine is used in the treatment of an autoimmunedisease or an inflammatory disorder. In some aspects, the combinationtherapy including but not limited to the combination of a TLR9 inhibitorand a vaccine is used in the treatment of an infectious disease.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as required. Ophthalmicformulation, ear drops, and eye drops are also contemplated as beingwithin the scope of this invention. Additionally, the present inventioncontemplates the use of transdermal patches, which have the addedadvantage of providing controlled delivery of a compound to the body.Such dosage forms can be made by dissolving or dispensing the compoundin the proper medium. Absorption enhancers can also be used to increasethe flux of the compound across the skin. The rate can be controlled byeither providing a rate controlling membrane or by dispersing thecompound in a polymer matrix or gel.

The present invention furthermore relates to a method for treating asubject suffering from a TLR9 related disorder, comprising administeringto said subject an effective amount of a compound of formulae (I) to(VI), in a therapeutically effective amount.

The term “therapeutically effective amount”, as used herein, refers to adosage and duration of administration which is commonly known in the artand recognized and utilized by the medical community. Such an amountwill vary depending on the particular agent(s) administered, the sizeand/or condition of the subject receiving treatment or other medicalfactors determined by the administering physician.

The compounds of the present invention are useful as anticancer agentsfor cancers that are responsive to TLR9 activation. In certainembodiments, the cancers include, but are not limited to cancer of thebreast, bladder, bone, brain, central and peripheral nervous system,colon, sarcoma, ovary, pancreas, prostate, rectum, renal, smallintestine, soft tissue, testis, stomach, skin, ureter, vagina and vulva;inherited cancers, retinomblastoma, Wilms tumor, leukemia, lymphoma,non-Hodgkins disease, chronic and acute myeloid leukaemia, acutelymphoblastic leukemia, Hodgkin's disease, multiple myeloma, and T-celllymphoma, myelodysplastic syndrome, and AIDS related cancer typediseases.

Immune suppression and/or inhibition according to the methods describedherein may be practiced on individuals including those suffering from adisorder associated with an unwanted activation of an immune response.The present disclosure also provides methods for inhibiting a TLR9induced response (e.g., in vitro or in vivo). In some variations, thecell is contacted with the TLR9 inhibitor in an amount effective toinhibit a response from the cell that contributes to an immune response.

Inhibition of TLR9 can be useful for treating and/or preventing avariety of diseases or disorders associated with cytokine activity.Conditions for which TLR9 inhibitors may be used as treatments include,but are not limited to, autoimmune diseases and inflammatory disorders.

Provided herein are methods of inhibiting an immune response in asubject, the method comprising administering to the individual at leastone TLR9 inhibitor as disclosed herein in an amount effective to inhibitthe immune response in the individual. In some variations, the immuneresponse is associated with an autoimmune disease. In further aspects,wherein inhibiting the immune response ameliorates one or more symptomsof the autoimmune disease. In still further aspects, wherein inhibitingthe immune response treats the autoimmune disease. In yet furtheraspects, wherein inhibiting the immune response prevents or delaysdevelopment of the autoimmune disease. In some variations, the TLRinhibitor inhibits a TLR9-dependent immune response. In some aspects, atleast one TLR inhibitor is administered in an amount effective toinhibit an immune response in the individual.

Provided herein are also methods of treating or preventing an autoimmunedisease in an individual, comprising administering to the individual aneffective amount of a TLR9 inhibitor. In some aspects, the autoimmunedisease is associated with the skin, muscle tissue, and/or connectivetissue. In some embodiments, the autoimmune disease is not evidenced inthe individual by skin, muscle tissue, and/or connective tissuesymptoms.

In some embodiments, the autoimmune disease is systemic. Autoimmunediseases include, without limitation, rheumatoid arthritis, autoimmunepancreatitis, systemic lupus erythematosus, type I diabetes mellitus,multiple sclerosis, antiphospholipid syndrome, sclerosing cholangitis,systemic onset arthritis, irritable bowel disease, scleroderma,Sjogren's disease, vitiligo, polymyositis, pemphigus vulgaris, pemphigusfoliaceus, inflammatory bowel disease including Crohn's disease,ulcerative colitis, and autoimmune hepatitis.

Accordingly, the invention provides a method of inhibiting TLR9 in ananimal, especially a mammal, preferably a human comprising administeringan effective amount of a compound of Formulae I-VI to the animal. Aswith all compositions for inhibition of an immune response, theeffective amounts and method of administration of the particular TLR9inhibitor formulation can vary based on the individual, what conditionis to be treated and other factors evident to one skilled in the art. Aneffective amount of a compound will vary according to factors known inthe art but is expected to be a dose of about 0.1 to 10 mg/kg, 0.5 to 10mg/kg, 1 to 10 mg/kg, 0.1 to 20 mg/kg, 0.1 to 20 mg/kg, or 1 to 20mg/kg.

In some embodiments, the combination therapy including but not limitedto the combination of a TLR9 inhibitor and a corticosteroid is used inthe treatment of an autoimmune disease or an inflammatory disorder. Insome embodiments, the autoimmune disease is selected from but notlimited to rheumatoid arthritis, systemic lupus erythematosus,autoimmune skin disease, multiple sclerosis, pancreatitis,glomerulonephritis, pyelitis, Sclerosing cholangitis, and type Idiabetes. In some embodiments, the autoimmune disease is Sjogren'sdisease.

Also provided herein are kits comprising a TLR9 inhibitor as providedherein, and instructions for use in the methods of inhibiting aTLR9-dependent immune response.

The kits may comprise one or more containers comprising a TLR inhibitor(or a formulation comprising a TLR inhibitor) as described herein, and aset of instructions, generally written instructions although electronicstorage media (e.g., magnetic diskette or optical disk) containinginstructions are also acceptable, relating to the use and dosage of theTLR inhibitor or formulation for the intended treatment (e.g.,suppression of a TLR9-dependent immune response, ameliorating one ormore symptoms of an autoimmune disease, ameliorating a symptom ofchronic inflammatory disease, decreasing cytokine production in responseto a virus, and/or treating and/or preventing one or more symptoms of adisease or disorder mediated by TLR9). The instructions included withthe kit generally include information as to dosage, dosing schedule, androute of administration for the intended treatment. The containers forthe TLR inhibitor (or formulations comprising a TLR inhibitor) may beunit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses.The kits may further comprise a container comprising an adjuvant.

The invention will be further explained with examples which are intendedto illustrate the particular embodiments and not to limit the scope ofthe invention.

As depicted in the Examples below, in certain exemplary embodiments,compounds are prepared according to the following general procedures. Itwill be appreciated that, although the general methods depict thesynthesis of certain compounds of the present invention, the followinggeneral methods, and other methods known to one of ordinary skill in theart, can be applied to all compounds and subclasses and species of eachof these compounds, as described herein.

The symbols and conventions used in the following descriptions ofprocesses, schemes, and examples are consistent with those used in thecontemporary scientific literature, for example, the Journal of theAmerican Chemical Society or the Journal of Biological Chemistry. Unlessotherwise indicated, all temperatures are expressed in ° C. (degreesCentigrade).

All solvents used were commercially available and were used withoutfurther purification. Reactions were typically run using anhydroussolvents. Flash column chromatography was generally carried out usingsymmetry C18 columns feature trifunctionally bonded C18 ligands on ahigh purity base-deactived silica.

All NMR spectra were recorded on Bruker DPX-400 NMR spectrometers(400.13 MHz). 1H-NMR chemical shifts (δH) are quoted in parts permillion (ppm) downfield from residual non-deuterated solvent peaks as areference signal, as per published guidelines (J. Org. Chem., Vol. 62,No. 21, 1997). Abbreviations for NMR data are s (singlet), d (doublet),t (triplet), q (quartet), m (multiplet).

High resolution LC-MS spectra were registered on API 165 EXspectrometer, equipped with Shimadzu LC10 Avp chromatographer andUV-detector Shimadzu SPD 10A vp, light scattering detector ELSD Sedex75, autosampler Gilson 215.

In general, the compounds of this invention can be prepared from readilyavailable starting materials. If such starting materials are notcommercially available, they may be prepared by standard synthetictechniques.

Analytical data of the compounds are summarized in the Table 1 below.

Compound Physical properties 1

NMR ¹H (DMSO D6): 7.75 (s, 1H), 7.55 (d, 2H), 6.15 (m, 6H), 3.80 (d,2H), 3.50 (s, 2H), 3.30 (s, 12H), 3.20 (q, 2H), 2.80 (m, 4H), 2.95 (s,2H), 2.85 (t, 2H), 2.35 (m, 1H), 1.75 (m, 6H). Mass m/z = 403 [M + H]⁺Yield 92% INS020_001 2

NMR ¹H (DMSO D6): 8.35 (s, 1H), 7.85 (t, 1H), 7.75 (d, 1H), 7.1 (m, 3H),6.90 (d, 1H), 4.35 (d, 2H), 3.55 (s, 2H), 3.30 (s, 5H), 3.25 (q, 2H),2.80 (t, 2H), 2.75 (m, 2H), 2.40-2.55(m, 4H), 1, 52 (m, 4H), 1.45 (q,2H) Mass m/z = 447 [M + H]⁺ Yield 96% 3

NMR ¹H (DMSO D6): 8.4 (s, 1H), 7.8 (t, 2H), 7, 56 (d, 2H), 6.95-7.2 (m,4H), 6.85 (d, 1H), 4.40 (d, 2H), 3.95 (s, 2H), 3.30 (s, 4H), 3.20 (q,2H), 2.85 (t, 2H), 2.60 (m, 2H), 2, 45 (s, 1H), 2.35 (m, 3H), 1.51-1.55(m, 4H), 1.45 (q, 2H). Mass m/z = 404 [M + H]⁺ Yield 95% 4

NMR ¹H (DMSO D6): 7.8 (s, 1H), 7.55 (d, 2H), 7, 10 (m, 6H), 3.80 (t,2H), 3.50 (s, 2H), 3.30 (s, 12H), 2.80-3.20 (m, 6H), 2.75 (m, 2H), 2.48(m, 5H), 2.27 (m, 1H), 1.35- 1.80 (m, 6H). Mass m/z = 403 [M + H]⁺ Yield95% INS020_002 5

Mass m/z = 421 [M + H]⁺ Yield 90% 6

NMR ¹H (DMSO D6): 7.80 (s, 1H), 7.60 (d, 2H), 7.00 (m, 6H), 3.90 (d,2H), 3.55 (s, 2H), 3.30 (m, 10H), 2.60-2.90 (m, 6H), 2.50 (s, 4H), 2.45(m, 1H), 1.45-1.75 (m, 4H). Mass m/z = 389 [M + H]⁺ Yield 91% 7

NMR ¹H (DMSO D6): 8.30 (s, 1H), 7.55 (m, 2H), 7.05 (m, 4H), 6.95 (d,1H), 3.60 (s, 2H), 3.25 (m, 1H), 2.90 (t, 2H), 2.75 (d, 2H), 2.65 (m,2H), 2.45-2.60 (m, 4H), 1.75 (m, 2H), 1.50 (m, 2H). Mass m/z = 433 [M +H]⁺ Yield 93% 8

NMR ¹H (DMSO D6): 8.45 (s, 1H), 7.60 (m, 2H), 7.20 (m, 4H), 6.80 (d,2H), 4.80 (d, 2H), 3.25 (m, 2H), 3.22 (t, 6H), 2.80 (t, 2H), 2.75 (m,2H), 2.65 (m, 2H), 2.50(m, 3H), 1.65 (m, 2H), 1.50 (m, 2H). Mass m/z =390 [M + H]⁺ Yield 93% 9

Mass m/z = 390 [M + H]⁺ Yield 94% INS020_003 10

NMR ¹H (DMSO D6): 7.85 (s, 1H), 7.65 (d, 1H), 7.48 (m, 1H), 7.10 (m,4H), 3.60 (m, 4H), 3.30 (m, 6H), 2.85 (t, 1H), 2.65 (m, 2H), 2.55 (m,4H), 1.75 (m, 2H), 1.55 (m, 2H), 1.45 (m, 1H). Mass m/z = 407 [M + H]⁺Yield 97% 11

NMR ¹H (DMSO D6): 7.85 (t, 1H), 7.65 (d, 2H), 6.90 (m, 4H), 6.50 (m,2H), 3.90 (d, 2H), 3.25 (m, 10H), 3.15 (m, 2H), 2.85 (m, 4H), 2.55 (s,2H), 2.35 (m, 1H), 1.65 (m, 2H), 1.45 (m, 2H). Mass m/z = 375 [M + H]⁺Yield 91% 12

NMR ¹H (DMSO D6): 8.38 (s, 1H), 7.95 (t, 1H), 7.75 (d, 1H), 6.95 (m,3H), 6.45 (m, 2H), 4.45 (d, 2H), 3.30 (m, 10H), 3.10 (m, 2H), 2.85 (m,4H), 2.45 (m, 3H), 2.65 (m, 2H), 1.45 (m, 2H). Mass m/z = 419 [M + H]⁺Yield 90% 13

NMR ¹H (DMSO D6): 8.45 (s, 1H), 7.95 (m, 1H), 7.77 (d, 1H), 6.90 (m,2H), 6.50 (m, 2H), 4.45 (d, 2H), 3.25 (m, 12H), 2.80-3.20 (m, 6H), 2.45(s, 1H), 2.40 (m, 1H), 1.75 (m, 2H), 1.45 (m, 2H). Mass m/z = 376 [M +H]⁺ Yield 92% 14

NMR ¹H (DMSO D6): 8.00 (m, 1H), 7.55 (d, 2H), 7.00 (m, 4H), 6.50 (m,2H), 3.85 (m, 2H), 3.80 (m, 9H), 3.10 (m, 2H), 2.85 (m, 4H), 2.50 (m,1H), 2.30 (m, 1H), 1.75 (m, 1H), 1.65 (m, 2H), 1.40 (m, 1H). Mass m/z =375 [M + H]⁺ Yield 92% 15

Mass m/z = 393 [M + H]⁺ Yield 96% INS020_004 16

NMR ¹H (DMSO D6): 7.88 (m, 1H), 7.55 (d, 2H), 6.90 (m, 4H), 6.50 (m,2H), 4.90 (m, 2H), 3.25 (m, 8H), 3.10 (m, 4H), 2.85 (m, 4H), 2.50 (m,1H), 2.35 (m, 1H), 1.70 (m, 6H). Mass m/z = 386 [M + H]⁺ Yield 98% 17

NMR ¹H (DMSO D6): 8.37 (s, 1H), 7.85 (m, 2H), 7.95 (m, 3H), 6.60 (t,1H), 6.45(m, 1H), 4.45 (d, 2H), 3.25 (m, 6H), 3.10 (m, 2H), 2.85 (m,6H), 2.45 (m, 2H), 1.65 (m, 4H), 1.45 (m, 2H). Mass m/z = 433 [M + H]⁺Yield 96% 18

NMR ¹H (DMSO D6): 8.45 (s, 1H), 7.90 (t, 1H), 7.75 (m, 1H), 6.95 (m,3H), 6.45(m, 2H), 3.30 (m, 5H), 3.10 (m, 6H), 2.85 (m, 2H), 2.45 (m,2H), 1.65 (m, 6H). Mass m/z = 390 [M + H]⁺ Yield 95% 19

NMR ¹H (DMSO D6): 7.95 (s, 1H), 7.55 (d, 2H), 7.95 (m, 4H), 3.95 (t,2H), 3.80 (m, 8H), 3.30 (m, 5H), 3.20 (m, 2H), 3.00 (m, 3H), 2.85 (m,2H), 2.55 (s, 1H), 2.35 (m, 1H), 1.90 (m, 1H), 1.79 (m, 4H), 1.45 (m,1H). Mass m/z = 389 [M + H]⁺ Yield 95% 20

NMR ¹H (DMSO D6): 7.95 (s, 1H), 7.60 (d, 1H), 7.55 (d, 1H), 7.20 (t,1H), 6.90 (m, 2H), 6.60 (t, 1H), 6.45 (d, 1H), 3.55 (t, 2H), 3.25 (m,10H), 3.10 (m, 2H), 2.90 (m, 2H), 2.80 (m, 4H), 1.50-1.80 (m, 6H), 1.45(m, 1H). Mass m/z = 407 [M + H]⁺ Yield 92% 21

NMR ¹H (DMSO D6): 7.75 (s, 1H), 7.60 (d, 1H), 6.90 (m, 3H), 6.65 (m,2H), 3.90 (d, 2H), 3.70 (s, 3H), 3.40 (m, 4H), 3.20 (m, 2H), 2.90 (t,2H), 2.75 (m, 2H), 2.55 (m, 2H), 2.30 (m, 5H), 1.70 (m, 2H), 1.60 (m,4H). Mass m/z = 419 [M + H]⁺ Yield 95% 22

NMR ¹H (DMSO D6): 8.35 (s, 1H), 7 75 (m, 2H), 6.90 (m, 2H), 3.70 (s,2H), 3.50 (s, 2H), 3.25 (m, 6H), 2.95 (t, 2H), 2.75 (m, 2H), 2.55 (m,2H), 2.45 (m, 4H), 1.70 (m, 2H), 1.50 (m, 2H). Mass m/z = 463 [M + H]⁺Yield 93% 23

NMR ¹H (DMSO D6): 8.40 (s, 1H), 7.75 (m, 2H), 6.90 (m, 2H), 6.35 (m,2H), 4.35 (d, 2H), 3.70 (s, 3H), 3.45 (t, 2H), 3.25 (s, 6H), 2.95 (m,2H), 2.75 (m, 2H), 2.45 (m, 4H), 1.50 (m, 4H), 1.75 (m, 2H), 1.45 (m,2H). Mass m/z = 420 [M + H]⁺ 24

Mass m/z = 419 [M + H]⁺ Yield 96% 25

Mass m/z = 437 [M + H]⁺ Yield 90% INS020_005 26

NMR ¹H (DMSO D6): 7.80 (t, 1H), 7.60 (d, 2H), 6.95 (m, 32H), 6.65 (m,2H), 3.85 (d, 2H), 3.65 (s, 3H), 3.10 (q, 2H), 2.90 (t, 2H), 2.70 (m,2H), 2.60 (m, 2H), 2.50 (m, 2H), 2.40 (m, 2H), 2.30 (m, 2H), 1.60 (m,6H). Mass m/z = 433 [M + H]⁺ Yield 91% 27

Mass m/z = 434 [M + H]⁺ Yield 95% 28

NMR ¹H (DMSO D6): 8.40 (s, 1H), 7.80 (m, 2H), 6.80 (t, 2H), 6.60 (m,2H), 4.40 (d, 2H), 3.70 (s, 3H), 3.40 (s, 1H), 3.30 (s, 4H), 3.10 (q,2H), 2.90 (t, 2H), 2.78 (m, 2H), 2.65 (m, 2H), 2.50 (m, 1H), 2.30 (m,2H), 1.60 (m, 6H). Mass m/z = 434 [M + H]⁺ Yield 96% 29

NMR ¹H (DMSO D6): 7.90 (s, 1H), 7.45 (d, 2H), 7.25 (s, 1H), 6.90 (m,1H), 6.70 (d, 2H), 6.65 (m, 1H), 6.60 (s, 1H), 3.55 (m, 7H), 3.45 (m,2H), 2.75 (m, 8H), 2.45 (m, 1H), 1.75 (m, 3H), 2.65 (m 2H), 2.40 (m,2H), 1.45 (m, 3H). Mass m/z = 433 [M + H]⁺ Yield 97% 30

NMR ¹H (DMSO D6): 7.90 (s, 1H), 7.70 (d, 1H), 7.55 (d, 1H), 7.15 (t,1H), 6.90 (d, 1H), 6.15 (m, 2H), 3.65 (s, 3H), 3.45 (m, 3H), 3.30 (m,6H), 3.10 (m, 2H), 3.80 (m, 1H), 2.75 (s, 2H), 2.65 (m 2H), 2.40 (m,2H), 1.65 (m, 6H). Mass m/z = 451 [M + H]⁺ Yield 90%

In some embodiments, the TLR9 inhibitors are selected from compounds 1,4, 9, 15, and 25, or from a compound having similar substitutionpatterns. In some embodiments, the TLR9 inhibitors are selected fromonly compounds 1, 4, 9, 15, and 25.

In some embodiments, the TLR9 inhibitor is compound 1 (i.e.,INS020_001).

In some embodiments, the TLR9 inhibitor is compound 4 (i.e.,INS020_002).

In some embodiments, the TLR9 inhibitor is compound 9 (i.e.,INS020_003).

In some embodiments, the TLR9 inhibitor is compound 15 (i.e.,INS020_004).

In some embodiments, the TLR9 inhibitor is compound 25 (i.e.,INS020_005).

Example 1

The compounds of the invention can be prepared using the followingprocedure.

Compound 1:1-(4-Cyanophenyl)-N-(3-(3,4-dihydroisoquinolin-2(19)-yl)propyl)piperidine-4-carboxamide

Method A.1-(4-Cyanophenyl)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)propyl)piperidine-4-carboxamide

To a solution of 1-(4-cyanophenyl)piperidine-4-carboxylic acid (1 mmol,230 mg) in DMF (2 mL) CDI (1 mmol, 162 mg) was added, and the mixturewas stirred at ambient temperature for 1 h, then3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-1-amine (1 mmol, 190.28 mg)was added. The mixture was stirred at ambient temperature overnight. Thesolvent was removed under reduced pressure, diluted with dichloromethaneand washed with 5% aqueous solution Na₂CO₃. Layers were separated, theorganic one dried over Na₂SO₄, filtered and concentrated under reducedpressure. The obtained residue was purified by column chromatography onsilica gel to yield1-(4-Cyanophenyl)-N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)propyl)piperidine-4-carboxamideas white solid (369 mg, 92%). MS: m/z=403 [M+H]⁺.

Compound 2:N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)propyl)-1-(4-(trifluoromethyl)phenyl)piperidine-4-carboxamide

Method B.N-(3-(3,4-dihydroisoquinolin-2(1H)-yl)propyl)-1-(5-(trifluoromethyl)pyridin-2-yl)piperidine-4-carboxamide

To a solution of1-(5-(trifluoromethyl)pyridin-2-yl)piperidine-4-carboxylic acid (1 mmol,274 mg) in DMF (2 mL) CDI (1 mmol, 162 mg) was added, and the mixturewas stirred at ambient temperature for 1 h, then3-(3,4-dihydroisoquinolin-2(1H)-yl)propan-1-amine (1 mmol, 190.28 mg)was added. The mixture was stirred at ambient temperature overnight. Thesolvent was removed under reduced pressure, diluted with dichloromethaneand washed with 5% aqueous solution Na₂CO₃. Layers were separated, theorganic one dried over Na₂SO₄, filtered and concentrated under reducedpressure. The obtained residue was purified by column chromatography onsilica gel to yieldN-(3-(3,4-dihydroisoquinolin-2(1H)-yl)propyl)-1-(5-(trifluoromethyl)pyridin-2-yl)piperidine-4-carboxamideas white solid (428 mg, 96%). MS: m/z=447 [M+H]⁺.

Compound 8:1-(5-cyanopyridin-2-yl)-N-(2-(3,4-dihydroisoquinolin-2(1H)-yl)ethyl)piperidine-4-carboxamide

Method C.1-(5-cyanopyridin-2-yl)-N-(2-(3,4-dihydroisoquinolin-2(1H)-yl)ethyl)piperidine-4-carboxamide

To a solution of 1-(5-cyanopyridin-2-yl)piperidine-4-carboxylic acid (1mmol, 231 mg) in DMF (2 mL) CDI (1 mmol, 162 mg) was added, and themixture was stirred at ambient temperature for 1 h, then-(3,4-dihydroisoquinolin-2(1H)-yl)ethanamine (1 mmol, 176.26 mg) wasadded. The mixture was stirred at ambient temperature overnight. Thesolvent was removed under reduced pressure, diluted with dichloromethaneand washed with 5% aqueous solution Na₂CO₃. Layers were separated, theorganic one dried over Na₂SO₄, filtered and concentrated under reducedpressure. The obtained residue was purified by column chromatography onsilica gel to yield1-(5-cyanopyridin-2-yl)-N-(2-(3,4-dihydroisoquinolin-2(1H)-yl)ethyl)piperidine-4-carboxamideas white solid (362 mg, 93%). MS: m/z=390 [M+H]⁺.

Compound 10:1-(4-cyano-2-fluorophenyl)-N-(2-(3,4-dihydroisoquinolin-2(1H)-yl)ethyl)piperidine-3-carboxamide

Method D:1-(4-cyano-2-fluorophenyl)-N-(2-(3,4-dihydroisoquinolin-2(1H)-yl)ethyl)piperidine-3-carboxamide

To a solution of 1-(4-cyano-2-fluorophenyl)piperidine-3-carboxylic acid(1 mmol, 248 mg) in DMF (2 mL) CDI (1 mmol, 162 mg) was added, and themixture was stirred at ambient temperature for 1 h, then-(3,4-dihydroisoquinolin-2(1H)-yl)ethanamine (1 mmol, 176 mg) was added.The mixture was stirred at ambient temperature overnight. The solventwas removed under reduced pressure, diluted with dichloromethane andwashed with 5% aqueous solution Na₂CO₃. Layers were separated, theorganic one dried over Na₂SO₄, filtered and concentrated under reducedpressure. The obtained residue was purified by column chromatography onsilica gel to yield1-(4-cyano-2-fluorophenyl)-N-(2-(3,4-dihydroisoquinolin-2(1H)-yl)ethyl)piperidine-3-carboxamideas yellowish solid (394 mg, 97%). MS: m/z=407 [M+H]⁺.

Compound 11.1-(4-cyanophenyl)-N-(2-(indolin-1-yl)ethyl)piperidine-4-carboxamide

Method E:1-(4-cyanophenyl)-N-(2-(indolin-1-yl)ethyl)piperidine-4-carboxamide

To a solution of 1-(4-cyanophenyl)piperidine-4-carboxylic acid (1 mmol,230 mg) in DMF (2 mL) CDI (1 mmol, 162 mg) was added, and the mixturewas stirred at ambient temperature for 1 h, then2-(indolin-1-yl)ethanamine (1 mmol, 162 mg) was added. The mixture wasstirred at ambient temperature overnight. The solvent was removed underreduced pressure, diluted with dichloromethane and washed with 5%aqueous solution Na₂CO₃. Layers were separated, the organic one driedover Na₂SO₄, filtered and concentrated under reduced pressure. Theobtained residue was purified by column chromatography on silica gel toyield1-(4-cyanophenyl)-N-(2-(indolin-1-yl)ethyl)piperidine-4-carboxamide aswhite solid (340 mg, 91%). MS: m/z=375 [M+H]⁺.

Compound 17:N-(3-(indolin-1-yl)propyl)-1-(5-(trifluoromethyl)pyridin-2-yl)piperidine-4-carboxamide

Method F:N-(3-(indolin-1-yl)propyl)-1-(5-(trifluoromethyl)pyridin-2-yl)piperidine-4-carboxamide

To a solution of1-(5-(trifluoromethyl)pyridin-2-yl)piperidine-4-carboxylic acid (1 mmol,274 mg) in DMF (2 mL) CDI (1 mmol, 162 mg) was added, and the mixturewas stirred at ambient temperature for 1 h, then3-(indolin-1-yl)propan-1-amine (1 mmol, 176 mg) was added. The mixturewas stirred at ambient temperature overnight. The solvent was removedunder reduced pressure, diluted with dichloromethane and washed with 5%aqueous solution Na₂CO₃. Layers were separated, the organic one driedover Na₂SO₄, filtered and concentrated under reduced pressure. Theobtained residue was purified by column chromatography on silica gel toyieldN-(3-(indolin-1-yl)propyl)-1-(5-(trifluoromethyl)pyridin-2-yl)piperidine-4-carboxamideas white solid (414 mg, 96%). MS: m/z=433 [M+H]⁺.

Intermediate 1: 2-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanamine

Method G

To a solution of 2-(2-hydroxyethyl)isoindoline-1,3-dione (10 mmol, 1.91g) and DIPEA (10 mmol, 1.29 g) in CH₂Cl₂, trifluoromethanesulfonicanhydride (1.15 eq, 3.25 g) dropwise were added at 5° C. The mixture wasstirred at room temperature for 1 h. After this time, the mixture waswashed with 5% aqueous solution of NaHCO₃ and water. Layers wereseparated, the organic one dried over Na₂SO₄ and evaporated in vacuo toyield 2-(1,3-dioxoisoindolin-2-yl)ethyl trifluoromethanesulfonate asyellow oil (2.94 g, 92%). MS: m/z=324 [M+H]⁺.

Method H

To a solution of 2-(1,3-dioxoisoindolin-2-yl)ethyltrifluoromethanesulfonate (5 mmol, 1.62 g) in CH₂Cl₂ (100 ml)6-methoxy-1,2,3,4-tetrahydroisoquinoline (0.8 eq, 0.65 g) and saturatedsolution of NaHCO₃ in water (100 ml) were added. The mixture was stirredat room temperature for 12 h. After this time, the mixture was washedwith water. Layers were separated, the organic one dried over Na₂SO₄ andevaporate to give crude product of2-(2-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)isoindoline-1,3-dione.

Method I

To a solution of crude2-(2-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)isoindoline-1,3-dionein MeOH (200 ml) N₂H₄.H₂O (25 mmol, 1.71 g) was added. The mixture wasstirred at room temperature for 12 h. A pellet developed gradually andwas filtered. The solvent was evaporated. The obtained residue waspurified by column chromatography on silica gel to give2-(6-methoxy-3,4-dihydroisoquinolin-2(M)-yl)ethanamine as white solid(0.93 g, 90%). MS: m/z=207 [M+H]⁺

Compound 21:1-(4-cyanophenyl)-N-(2-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)piperidine-4-carboxamide

Method J:1-(4-cyanophenyl)-N-(2-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)piperidine-4-carboxamide

To a solution of 1-(4-cyanophenyl)piperidine-4-carboxylic acid (1 mmol,230 mg) in DMF (2 mL) CDI (1 mmol, 162 mg) was added, and the mixturewas stirred at ambient temperature for 1 h, then2-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanamine (1 mmol, 206 mg)was added. The mixture was stirred at ambient temperature overnight. Thesolvent was removed under reduced pressure, diluted with dichloromethaneand washed with 5% aqueous solution Na₂CO₃. Layers were separated, theorganic one dried over Na₂SO₄, filtered and concentrated under reducedpressure. The obtained residue was purified by column chromatography onsilica gel to yield1-(4-cyanophenyl)-N-(2-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)piperidine-4-carboxamideas white solid (397 mg, 95%). MS: m/z=419 [M+H]⁺.

Compound 22:N-(2-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1-(5-(trifluoromethyl)pyridin-2-yl)piperidine-4-carboxamide

Method K:N-(2-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1-(5-(trifluoromethyl)pyridin-2-yl)piperidine-4-carboxamide

To a solution of1-(5-(trifluoromethyl)pyridin-2-yl)piperidine-4-carboxylic acid (1 mmol,274 mg) in DMF (2 mL) CDI (1 mmol, 162 mg) was added, and the mixturewas stirred at ambient temperature for 1 h, then2-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethanamine (1 mmol, 206 mg)was added. The mixture was stirred at ambient temperature overnight. Thesolvent was removed under reduced pressure, diluted with dichloromethaneand washed with 5% aqueous solution Na₂CO₃. Layers were separated, theorganic one dried over Na₂SO₄, filtered and concentrated under reducedpressure. The obtained residue was purified by column chromatography onsilica gel to yieldN-(2-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)ethyl)-1-(5-(trifluoromethyl)pyridin-2-yl)piperidine-4-carboxamideas white solid (430 mg, 93%). MS: m/z=463 [M+H]⁺.

Intermediate 2:3-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)propan-1-amine

Method L

6-Methoxy-1,2,3,4-tetrahydroisoquinoline hydrochloride (10 mmol, 1.99 g)was dissolved in ethanol (100 mL) and treated with Et₃N (10 mmol, 1.01g) and an access of acrylonitrile (30 mmol, 1.59 g). The mixture washeated to reflux for 5 h. The volatiles were removed under reducedpressure, and the residue was portioned between CH₂Cl₂ and water. Theorganic layer was washed with brine, dried (Na₂SO₄), and filtered.Evaporation of the solvent yielded the crude product, which could bepurified by chromatography to give3-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)propanenitrile as whitesolid (2.07 g, 96%). MS: m/z=217 [M+H]⁺.

Method M

A solution of3-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)propanenitrile (5 mmol,1.08 g) in THF (10 ml) was added dropwise to a freshly preparedsuspension of AlCl₃ (9 mmol, 1.20 g) and LiAlH₄ (9.25 mmol, 0.35 g) inTHF (dry, 250 mL) under nitrogen atmosphere. The mixture was allowed tostir at room temperature overnight. Workup was initiated by carefulsubsequent addition of water (5 mmol, 0.1 mL), NaOH (1N, 5 mL), andanother portion of water (20 mmol, 0.4 mL) and filtration of the saltsthus formed over Celite. The clear solution was dried (Na₂SO₄) andconcentrated in vacuo to yield3-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)propan-1-amine as whitesolid (0.94 g, 86%). MS: m/z=221 [M+H]⁺.

Compound 29:1-(4-cyanophenyl)-N-(3-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)piperidine-3-carboxamide

Method N:1-(4-cyanophenyl)-N-(3-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)piperidine-3-carboxamide

To a solution of 1-(4-cyanophenyl)piperidine-3-carboxylic acid (1 mmol,230 mg) in DMF (2 mL) CDI (1 mmol, 162 mg) was added, and the mixturewas stirred at ambient temperature for 1 h, then3-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)propan-1-amine (1 mmol, 220mg) was added. The mixture was stirred at ambient temperature overnight.The solvent was removed under reduced pressure, diluted withdichloromethane and washed with 5% aqueous solution Na₂CO₃. Layers wereseparated, the organic one dried over Na₂SO₄, filtered and concentratedunder reduced pressure. The obtained residue was purified by columnchromatography on silica gel to yield1-(4-cyanophenyl)-N-(3-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)piperidine-3-carboxamideas white solid (419 mg, 97%). MS: m/z=433 [M+H]⁺.

Compound 30:1-(4-cyano-2-fluorophenyl)-N-(3-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)piperidine-3-carboxamide

Method O:1-(4-cyano-2-fluorophenyl)-N-(3-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)piperidine-3-carboxamide

To a solution of 1-(4-cyano-2-fluorophenyl)piperidine-3-carboxylic acid(1 mmol, 248 mg) in DMF (2 mL) CDI (1 mmol, 162 mg) was added, and themixture was stirred at ambient temperature for 1 h, then3-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)propan-1-amine (1 mmol, 220mg) was added. The mixture was stirred at ambient temperature overnight.The solvent was removed under reduced pressure, diluted withdichloromethane and washed with 5% aqueous solution Na₂CO₃. Layers wereseparated, the organic one dried over Na₂SO₄, filtered and concentratedunder reduced pressure. The obtained residue was purified by columnchromatography on silica gel to yield1-(4-cyano-2-fluorophenyl)-N-(3-(6-methoxy-3,4-dihydroisoquinolin-2(1H)-yl)propyl)piperidine-3-carboxamideas white solid (405 mg, 90%). MS: m/z=451 [M+H]⁺.

Example 2

Select compounds were used for screening as agonists for human TLR2,TLR3, TLR4, TLR5, TLR7, TLR8, or TLR9. The screened compounds include:compound 1 (i.e., INS020_001); compound 4 (i.e., INS020_002); compound 9(i.e., INS020_003); compound 15 (i.e., INS020_004); and compound 25(i.e., INS020_005). Each compound was screened for potentialantagonistic effect on various human receptors known to recognizepathogen associated molecular patterns (PAMPs).

Samples and controls are tested in duplicate on recombinant HEK-293 celllines. These cell lines functionally over express a given TLR protein aswell as a reporter gene which is a secreted alkaline phosphatase (SEAP).The production of this reporter gene is driven by a NF-KB induciblepromoter. The magnitude of activation is represented in optical densityvalues (OD). A recombinant HEK-293 cell line for the reporter gene onlywas used as a negative control for the TLR cell lines (mentioned as TLR−in the data). This negative control cell line does not over-express anyTLR gene, but the reporter gene only (alkaline phosphatase). Thisreporter gene is directly inducible with TNFα. The non-induced value foreach clone is the background signal of the cell line.

In a 96-well (200 μl total volume) containing the appropriate cells(50,000-80,000 cells/well), 20 μL of the test article is added to thewell and is incubated with the cells at 37° C. with 5% CO₂ for 3 hoursprior to the addition of the agonist control. After further incubationwith the agonist at 37° C. with 5% CO₂ for 16-24 hours, the opticaldensity (OD) is read at 630.

Following is the list of different ligands used as positive inductioncontrols in this study:

Receptor Agonist Ligand Final Concentration hTLR2 Pam2CSK4 1 ng/ml hTLR3Poly I:C (HMW) 1 μg/ml hTLR4 LPS-EK - Standard LPS from 10 ng/ml E. coliK12 hTLR5 FLA-ST - Standard flagellin from 1 μg/ml S. thyphimurium hTLR7R848 1 μg/ml hTLR8 TL8-506 1 μg/ml hTLR9 ODN 2006 10 μg/ml

The control cell line (TLR-) is activated by TNFα.

Compound(s) Tested:

Test article Weight Reconstitution volume INS020_001 2.29 mg/vial 2.84ml INS020_002 2.22 mg/vial 2.76 ml INS020_003 2.06 mg/vial 2.65 mlINS020_004 2.24 mg/vial 2.85 ml INS020_005 2.39 mg/vial 2.74 mlINS020_100 1.49 mg/vial 2.80 mlPreparation of Test Article:

Samples were reconstituted in DMSO with the volume noted above to obtaina 2 mM stock solution. Stock solution was further diluted with completeDMEM 20-fold to obtain a solution at 100 μM. 20 μl of the diluted sampleis used to treat the cells in a 200 μl of final reaction volume. Thesample has therefore been tested at 10 μl final concentration.

Activation of the SEAP reporter is detected as OD value. OD value issubtracted by average non-induced (NI) value. The NI subtractedduplicates are averaged and presented in a histogram format hereafter.The values in the tables are the corresponding OD values for the Agonistand the Compound (e.g., shown as compound number).

TLR TLR TLR2 TLR3 TLR4 TLR5 TLR7 TLR8 TLR9 Agonist 1.71 2.03 1.58 1.652.51 2.05 2.32 1.72 1 1.22 1.85 1.21 1.48 2.41 1.73 2.24 0.06 Agonist1.71 2.03 1.58 1.65 2.51 2.05 2.32 1.72 4 1.35 1.8 1.36 1.6 2.21 1.782.16 0.14 Agonist 1.71 2.03 1.58 1.65 2.51 2.05 2.32 1.72 9 1.25 2.021.37 1.54 2.16 1.73 2.17 0.32 Agonist 1.71 2.03 1.58 1.65 2.51 2.05 2.321.72 15  1.17 2.15 1.37 1.5 2.45 1.72 2.25 1.51 Agonist 1.71 2.03 1.581.65 2.51 2.05 2.32 1.72 25  1.12 2.04 1.35 1.47 2.41 1.73 2.28 0.34Agonist 1.71 2.03 1.58 1.65 2.51 2.05 2.32 1.72 Control 1.64 2.16 1.521.95 2.22 −0.01 0.01 0.10 TLR TLR2 TLR3 TLR4 TLR5 TLR7 TLR8 TLR9 Agonist100%  100% 100%  100%  100%  100%  100%  100%  1 71%  91% 77% 90% 96%84% 97%  4% 4 79%  88% 86% 97% 88% 87% 93%  8% 9 73%  99% 87% 93% 86%84% 94% 18% 15 68% 106% 87% 91% 98% 84% 97% 88% 25 65% 100% 86% 89% 96%84% 98% 20% Control 96% 106% 96% 118%  88%  0%  0%  6%

At 10 μM, test articles Compound 1, Compound 4, Compound 9, and Compound25 show specific and significant antagonistic effect on human TLR9. At10 μM, the control is shown to be a general agonist for TLR7, TLR8 andTLR9. Thus, specific TLR9 inhibitors that are selective for TLR9 overother TLRs include Compound 1, Compound 4, Compound 9, and Compound 25.

The invention claimed is:
 1. A method for inhibiting TLR9 activity, themethod comprising: administering a compound to a TLR9 in an amountsufficient to inhibit activity thereof, the compound having a structureof Formula (I), or a stereoisomeric form, a mixture of stereoisomericforms, or pharmaceutically acceptable salts thereof,

wherein, ring B is a substituted monocycle containing 3-7 atoms, themonocycle being selected from an aryl or heteroaryl, wherein theheteroaryl has from 1 to 4 heteroatoms, which are independently selectedfrom nitrogen, oxygen, and sulfur; G represents a substituted orunsubstituted C₀-C₅ alkylene; one of W, U, E and J represents CR-T andthe rest of W, U, and J are independently absent or independentlyrepresent CR₂ or NR; T represents:

wherein, Z is selected from —O—C(NR)— and —(NR)C—O—; X represents(—CH₂—)_(n) wherein n=1 to 12, thereby forming an alkylene chain,wherein the alkylene chain is optionally substituted with halogen,C₁-C₂₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, halogenated C₁-C₁₀ alkyl,hydroxy C₁-C₁₀ alkyl, or C₁-C₁₀alkoxy; A is 3 to 8 membered heterocyclylfused with an aryl or heteroaryl, which can be unsubstituted orsubstituted with one or more R substituents; R₁ is one or more of,independently of each other, H, halogen, halogenated C₁-C₁₀ alkyl,hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, or —CN; R₂ is one or more of,independently of each other, H, C₁-C₂₀ alkyl, halogenated C₁-C₂₀ alkyl,—OR, —SR, or —CN; and each R is independently H, C₁-C₂₀ alkyl, C₂-C₂₀alkenyl, C₂-C₂₀ alkynyl, hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy,halogenated C₁-C₂₀ alkyl, halogen, —OH, —NO₂, —CN, —COOH, —CHO, —SO₃H,—SO₂R, —SOR, —NH₂, —NHR, —NR₂, CHal₃, —NHCO(C₁-C₁₀)alkyl, —CONHR,—C(O)R, —CO₂R, —C(O)N(R)₂, —NRC(O)R, —NRC(O)N(R)₂, —NRSO₂R.
 2. Themethod according to claim 1, wherein the compound is administered to theTLR9 in vitro.
 3. The method according to claim 1, wherein the compoundis administered to the TLR9 in vivo.
 4. The method according to claim 1,wherein the compound is administered to a subject having the TLR9,wherein the subject is susceptible or has a disease or disorder mediatedby the TLR9.
 5. The method of claim 4, wherein the subject has aincludes at least one of: a disorder or disease associated with theover-stimulation of the subject's immune system by microbes;interferon-mediated diseases; or inflammatory cytokine-mediatedinflammation diseases.
 6. The method according to claim 5, furthercomprising treating the disorder or disease associated with theover-stimulation of the subject's immune system by microbes.
 7. Themethod according to claim 5, further comprising treating theinterferon-mediated disease.
 8. The method according to claim 5, furthercomprising treating the inflammatory cytokine-mediated inflammationdiseases.
 9. The method of claim 4, further comprising treating at leastone of: antiphospholipid syndrome, autoimmune hepatitis, autoimmunemyocarditis, autoimmune orchitis, autoimmune pancreatitis, autoimmuneretinopathy, rheumatoid arthritis, psoriatic arthritis, osteoarthritis,systemic Lupus Erythematosus, lupus nephritis, osteoporosis, systemicsclerosis, multiple sclerosis, psoriasis, diabetes, inflammatory boweldisease (Crohn's Disease and Ulcerative Colitis),Hyperimmunoglobulinemia D, periodic fever syndrome, systemic juvenileidiopathic arthritis, sepsis, atherosclerosis, Celiac disease, Sjogren'sSyndrome, Alzheimer's disease, Parkinson's disease, or cancer.
 10. Themethod of claim 9, wherein the cancer is selected from colorectalcancer, breast cancer, ovarian carcinoma, pancreatic cancer, lungcancer, renal cell carcinoma, cervical cancer and multiple myeloma. 11.The method of claim 1, further comprising inhibiting TLR9 activity withthe compound.
 12. The method of claim 4, further comprising inhibitinginflammation in the subject with the compound.
 13. The method of claim4, further comprising inhibiting activation of an immune system of thesubject with the compound.
 14. The method of claim 1, further comprisingcontacting cells with the compound, wherein the cells express the TLR9.15. The method of claim 1, further comprising: the compound having astructure of Formula (III), or a stereoisomeric form, a mixture ofstereoisomeric forms, or pharmaceutically acceptable salts thereof,

wherein, Y and L are independently CR or CH, one of W, U, E and Jrepresents CH(T) and the rest of W, U, and J are independently absent orindependently represent CH₂; T represents:

wherein, Z is selected from —O—C(NR)— and —(NR)C—O—; X represents(—CH₂—)_(n) wherein n=1 to 24, thereby forming an alkylene chain,wherein the alkylene chain is optionally substituted with halogen,C₁-C₂₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, halogenated C₁-C₁₀ alkyl,hydroxy C₁-C₁₀ alkyl, or C₁-C₁₀alkoxy; A represents:

any one of A1, A2, A3, or A4 is unsubstituted or substituted with one ormore R groups; R₁ is one or more of, independently of each other, H,halogen, halogenated C₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy,or —CN; R₂ is one or more of, independently of each other, H, C₁-C₂₀alkyl, halogenated C₁-C₂₀ alkyl, —OR, —SR, or —CN; and each R isindependently H, C₁-C₂₀ alkyl, C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, hydroxyC₁-C₁₀ alkyl, alkoxy, halogenated C₁-C₂₀ alkyl, halogen, —OH, —NO₂, —CN,—COOH, —CHO, —SO₃H, —SO₂R, —SOR, —NH₂, —NHR, —NR₂, CHal₃,—NHCO(C₁-C₁₀)alkyl (e.g., alkyl-amide), —CONHR, —C(O)R, —CO₂R,—C(O)N(R)₂, —NRC(O)R, —NRC(O)N(R)₂, —NRSO₂R.
 16. The method of claim 15,wherein: Y is CR and L is CH, R₁ is para position; one of W, U, E and Jrepresents CH(T) and the rest of W, U, and J are independently absent orindependently represent CH₂; X represents (—CH₂—)_(n) wherein n=1, 2, or3, thereby forming an alkylene chain; A represents:

A1 is unsubstituted or substituted with one or more R groups; R₁ is oneor more of, independently of each other, H, halogen, halogenated C₁-C₁₀alkyl, hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, or —CN; R₂ is one or moreof, independently of each other, H, C₁-C₂₀ alkyl, halogenated C₁-C₂₀alkyl, —OR, —SR, or —CN; and each R is independently H, C₁-C₂₀ alkyl,C₂-C₂₀ alkenyl, C₂-C₂₀ alkynyl, hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy,halogenated C₁-C₂₀ alkyl, halogen, —OH, —NO₂, —CN.
 17. The method ofclaim 1, the compound having a structure of Formula (IV), or astereoisomeric form, a mixture of stereoisomeric forms, orpharmaceutically acceptable salts thereof,

wherein, Y and L are independently CH or CR, wherein at least one of Yor L is CH; G1 is CH₂ or absent; each R is independently selected fromH, halogen, C₁-C₆ alkyl, halogenated C₁-C₆ alkyl, hydroxy C₁-C₆ alkyl,C₁-C₆ alkoxy, or —CN; and X represents (—CH₂—)_(n), wherein n=2 or 3,thereby forming an alkylene chain.
 18. The method of claim 17, thecompound having a structure of Formula (VI), or a stereoisomeric form, amixture of stereoisomeric forms, or pharmaceutically acceptable saltsthereof,

wherein, G1 is CH or absent; X represents (—CH₂—)_(n) wherein n=1 to 4,thereby forming an alkylene chain, the alkylene chain is optionallysubstituted with halogen, C₁-C₆ alkyl, C₂-C₈ alkenyl, C₃-C₈ cycloalkyl,C₂-C₈ alkynyl, halogenated C₁-C₆ alkyl, hydroxy C₁-C₆ alkyl, orC₁-C₆alkoxy; R3 is one or more of H, halogen, halogenated C₁-C₁₀ alkyl,hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy, or —CN; R4 is one or more of H,halogen, halogenated C₁-C₁₀ alkyl, hydroxy C₁-C₁₀ alkyl, C₁-C₁₀ alkoxy,or —CN; R5 is one or more of H, halogen, halogenated C₁-C₁₀ alkyl,hydroxy C₁-C₁₀ alkyl, or C₁-C₁₀ alkoxy.
 19. The method of claim 18,wherein: G1 is CH or absent; X represents (—CH₂—)n wherein n=2 or 3,thereby forming an alkylene chain; R³ is —CN; R⁴ is one or more of H,halogen, or halogenated C₁-C₁₀ alkyl; and R⁵ is one or more of H,hydroxy C₁-C₁₀ alkyl, or C₁-C₁₀ alkoxy.
 20. The method of claim 1,wherein the compound is selected from:


21. The method of claim 20, wherein the compound is selected from:Compound 1, Compound 4, Compound 9, or Compound 25.